Compounds for detecting and treating Mycoplasma hyopneumoniae

ABSTRACT

The present disclosure relates to aptamers, polynucleotides, and nuclei acid molecules, which include a polynucleotide sequence capable of specifically binding polypeptides participating in  M. hyopneumoniae  infection. Also provided are methods of using nucleic acid molecules, polynucleotides and synthetic antibodies directed there against for detection, treating and neutralization of  M. hyopneumoniae  infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/312,207 filed Dec. 20, 2018, now U.S. Pat. No. 11,396,655, whichclaims priority to Canadian Application No. 2,940,637 filed Aug. 30,2016, each of which hereby is incorporated by reference in itsrespective entirety.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety. The Sequence Listing was created on Mar. 1, 2022, has afile name of FAERO-P002-US1 Seq_Listing_ASCII.txt, and is 44 kilobytesin size.

FIELD

The present invention relates to aptamers, nucleic acid molecules,synthetic antibodies binding to M. hyopneumoniae, which can be utilizedfor detecting and treating M. hyopneumoniae infection, particularly inswines, and pharmaceutical composition containing same.

BACKGROUND

M. hyopneumoniae are bacteria known to cause the Porcine EnzooticPneumonia (PEP), a highly contagious and chronic disease affectingpigs^([1]). As with other mollicutes, M. hyopneumoniae bacterium issmall in size, (400-120) nm), has a small genome (893-920 kilo-basepairs (kb)) and lacks a cell wall^([1-2]). It is considered to bedifficult to grow in laboratories due to its complex nutritionalrequirements. This bacterium is a major concern in the livestockindustry as it causes a significant reduction in the growing weight ofpigs, and is a known potentiation vector for SIV^([3]), and M.hyopneumoniae ^([4]). Losses in the U.S.A. have been previouslyestimated at 400 million to 1 billion dollars per annum^([5]). Porcineenzootic pneumonia is endemic worldwide and M. hyopneumoniae is presentin almost every pig herd, and lesions of enzootic pneumonia areconsistently observed in >50% of swine at slaughter^([6]). Althoughreported prevalence varies according to different researchers,inspections carried out in several countries show that typical lesionsof M. hyopneumoniae is observed in 50-95% of slaughtered pigs, whilst60-99% of herds may be positive to M. hyopneumoniae ^([1]).

To date, vaccination strategies, which constitute the basis of M.hyopneumoniae control, have been directed at preventing morbidity andmortality. Control of M. hyopneumoniae infections, apart fromimprovements in husbandry and the environment, is primarily with the useof antimicrobial products, but they have limitations. Medicatedprotection only lasted during treatment and for a short periodafterwards such that M. hyopneumoniae is not completely eliminated andthe herd have not necessarily developed much immunity (Haesebrouck,⁸).This has led to the development of pulse medication and strategicmedication programs to prolong the medicinal protection or to target thestress periods when disease was likely to flare up.

Most antibiotics only treat M. hyopneumoniae and not the secondarybacteria so a variety of combinations of antibiotics are used to broadenthe control effect. In the case of outbreaks, antibiotics such astetracyclines, lincomycin or tiamulin, show low minimum inhibitoryconcentration (MIC) (i.e. the lowest concentration of an antimicrobialthat will inhibit the visible growth of a microorganism).Fluoroquinolones are also effective but, owing to current concernsregarding antimicrobial resistance, their use in pigs should belimited^([7, 8]). The efficacy of doxycycline has been demonstrated inthe treatment of porcine enzootic pneumonia (PEP).

Although the current antimicrobial medication and vaccination confersbeneficial effects in most infected herds, the effects are variablebetween herds. The variable results may be due to different factors suchas improper medication/vaccine storage conditions, different injectiontechniques, antigenic differences between field strains and vaccinestrains, presence of other diseases at the time oftreatment/vaccination, interferences of vaccine-induced immune responsesby maternally derived (colostral) antibodies, etc. Hence there is plentyof space to develop much better anti-microbial drugs.

Treatment of this disease is limited to antibiotics, which are currentlyineffective, as they do not completely remove the infection^([7]). Atbest, vaccines are found to reduce the severity of the disease but donot prevent the disease from occurring in non-infected pigs^([8]).

Strictly speaking, M. hyopneumoniae is a pathogen of swine and alternatehosts or intermediary vectors have not been found. All electronmicroscopy studies of infected lung tissues have shown that M.hyopneumoniae interacts almost exclusively with (i) cilia on theepithelial surfaces that lines the trachea, the bronchi and thebronchioles in the porcine upper respiratory tract, and with (ii)pulmonary alveolar macrophages (PAM)^([1, 9]). More specifically, thisbacterium is found attached along the entire length of the cilia butrarely to the epithelial cell body^([1-2]).

To survive and proliferate as an infectious agent, M. hyopneumoniae must(a) enter the respiratory tract of its host, (b) traverse mucous layers,(c) resist the mucociliary escalator, (d) adhere and colonize theepithelial cilia. (d) secure essential nutrients for growth andreplication, (f) evade immune response, and (g) repeat the cycle ofinfection by transmission to new hosts via airborne mucosal droplets.The colonization of the upper respiratory track results in thedestruction of the mucociliary escalator via ciliostasis (they causecilia to stop beating), loss of cilia and eventually epithelial celldeath; which is the source of the lesions found in the lungs of pigswith porcine enzootic pneumonia^([1]). The analysis of the literatureshows that the mechanisms being utilized by M. hyopneumoniae to colonizerespiratory cilia requires multiple adhesins and strategies to avoidimmune detection. For example, heparin effectively blocks the binding ofM. hyopneumoniae to porcine cilia indicating that M. hyopneumoniae isreliant on the presentation of heparin-binding proteins on its cellsurface. This is consistent with the fact that proteoglycans with highlysulphated glycosaminoglycan (GAG) side chains are prominently displayedon the surface of ciliated epithelium lining the porcine respiratorytract^([10]). This might also explain why the immune response to thepresence of M. hyopneumoniae is slow and ineffective^([7-8]).

As of April 2012, five separate strains (232, 7448, 7422, Perdue MasterSeed (PMS), and J) of this Mycoplasma have had their genomes sequenced,making it the most sequenced Mycoplasma ^([11]); the first three beingvirulent, the forth one mildly virulent, and the last one with novirulence. Current research is mainly focused on identifying the “rightadhesins” with a final goal of developing an effective vaccine thatprevents Mycoplasma hyopneumoniae from attaching to lung cilia.

SUMMARY

It would be advantageous to have improved compositions, which candiagnose and treat M. hyopneumoniae infection. Here, we describe ssDNAaptamers for M. hyopneumoniae. These were developed using SELEX methodand the binding domain of the P97 surface protein as the target ratherthan the entire Mycoplasma organism. The characterization of theaptamers and their use as an analytical tool for the detection of M.hyopneumoniae were evaluated using various enzyme-linkedantibody-aptamer assays (ELISA). According to one aspect, the aptamerswere then further modified to permit binding on various biosensorssurfaces for a more rapid and convenient detection of M. hyopneumoniae.

We disclose herein nucleic acid molecules comprising at least onepolynucleotide sequence capable of specifically binding various peptidiccomplexes participating in the M. hyopneumoniae infection, including aM. hyopneumoniae polypeptide or a host cell polypeptide.

According to an embodiment, we disclose a nucleic acid moleculecomprising at least one polynucleotide sequence capable of specificallybinding to: (i) a polypeptide expressed by M. hyopneumoniae; or (ii) ahost molecule expressed by a host organism; wherein the polypeptide orthe host molecule is involved in binding M. hyopneumoniae to the hostorganism.

The polynucleotide sequences are capable of:

a) binding a region of the P97_({768-1082}), P97_({768-1050}),P97_({768-925}), P116_({700-1010}), P102_({1-324}), or P102_({1-529})surface proteins of M. hyopneumoniae or a fragment thereof having atleast 80%, 85%, 90% or 95 identity with P977_({768-1082}),P97_({768-1050}), P97_({768-925}), P116_({700-1010}), P102_({1-324}), orP102_({1-529}) that control binding of the Mycoplasma onto the ciliaand/or pulmonary alveolar macrophages (PAM);b) binding to a surface protein of M. hyopneumoniae carrying a motif|AAKPE|AAKPV|AAKPE|TTKPV| or a motif that has at least an 80%, 85%, 90%,or 95% identity with said motif; orc) binding to P97, P102, P116, P146, P216, or P159 surface proteins ofM. hyopneumoniae or a fragment having at least 80%, 85%, 90%, or 95%sequence identity with the P97, P102, P116, P146, P216, and P159 surfaceproteins, that control binding of M. hyopneumoniae onto the cilia and/orpulmonary alveolar macrophages (PAM).

According to further features, the polynucleotide sequence is APT3Cooc10(SEQ ID NO:1), APT3Cooc4 (SEQ ID NO:2), APT3Cooc2 (SEQ ID NO:3),APT3Histo1 (SEQ ID NO:4), APT3Cooc3 (SEQ ID NO:5), APT3Histo2 (SEQ IDNO:6), APT3Histo4 (SEQ ID NO:7), APT3Histo5 (SEQ ID NO:8), APT3Histo9(SEQ ID NO:9), or APT3Cooc7 (SEQ ID NO:10) or is a sequence having atleast 60%, 65%, 75%, 80%, 85%, 9)%, or 95% identity to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

According to further features, the polynucleotide sequence is APT3Cooc10(SEQ ID NO:1), APT3Histo1 (SEQ ID NO:4), or APT3Cooc3 (SEQ ID NO:5).

According to still further features in the described embodiments thehost cell molecule are glycolipids found on the cilia of porcinepulmonary epithelium cells, such as GM3, La, Lb, or Lc. (Q. Zhang etal., Glycolipid receptors for attachment of Mycoplasma hyopneumoniae toporcine respiratory ciliated cells. Infection & Immunity, 1994, vol. 62,pp. 4367-4373. Also C. Hermans & A. Bernard, Lung epithelium-specificproteins: Characteristics and potential applications markers. Am. J. ofRespi. Crit. Care Med., 1999, vol. 159, pp. 646-678).

According to still further features in the described embodiments thepolynucleotide sequence is single stranded.

According to still further features in the described embodiments thepolynucleotide sequence is ssDNA.

According to still further features in the described embodiments thenucleic acid molecule further comprising a detectable label.

According to still further features in the described embodiments thepolynucleotide sequence include FDG ([¹⁸F]-2-fluoro-2-deoxy-D-glucose)and/or PEG modified nucleotides.

According to still further features in the described embodiments thepolynucleotide sequence have a length of 10 to 45 nucleotides.

According to still further features in the described embodiments thehost molecule is a monosialoganglioside-like receptor at the surface ofcilia of porcine pulmonary epithelium cells.

According to another aspect of the present invention there is provided amethod of isolating a molecule capable of inhibiting M. hyopneumoniaeinfection, the method comprising: (a) contacting a plurality of nucleicacid molecules with a polypeptide consisting of P97_({768-1082}),P97_({768-1050}), P97_({768-925}), P116_({700-1010}), P102_({1-324}), orP102_({1-529}); or with a polypeptide having at least 80%, 85%, 90% or95 identity with P97_({768-1082}) P97_({768-1050}), P97_({768-925}),P116_({700-1010}), P102_({1-324}), or P102_({1-529}); (b) identifying atleast one nucleic acid molecule from the plurality of nucleic acidmolecules capable of specifically binding the polypeptide; and (c)isolating the at least one nucleic acid molecule capable of binding thepolypeptide.

According to still further features in the described embodiments themethod further comprising generating the plurality of nucleic acidmolecules using a combinatorial synthesis approach prior to step (a).

According to still further features in the described embodiments themethod further comprising modifying the plurality of nucleic acidmolecules prior to step (a) or following step (c).

According to still further features in the described embodiments themethod further comprising repeating steps (a) to (c).

According to yet another aspect of the present invention there isprovided a pharmaceutical composition comprising a nucleic acid moleculeincluding a polynucleotide sequence capable of specifically binding apeptidic complex participating in M. hyopneumoniae infection of cells,in particular a nucleic acid molecule as described hereinbefore, and aphysiologically acceptable carrier.

According to a further aspect of the present invention there is provideda composition of matter, preferably a pharmaceutical composition,comprising an anti-microbial agent conjugated to a nucleic acid moleculeincluding a polynucleotide sequence capable of specifically binding apolypeptide participating M. hyopneumoniae infection of cells.

According to still another aspect of the present invention there isprovided an article-of-manufacture comprising packaging material and apharmaceutical composition identified for treating or preventing M.hyopneumoniae infection being contained within the packaging material,the pharmaceutical composition including, as an active ingredient, anucleic acid molecule including a polynucleotide sequence capable ofspecifically binding a peptide participating in M. hyopneumoniaeinfection of cells, in particular a pharmaceutical composition asdescribed hereinbefore.

According to still further features in the described embodiments thepharmaceutical composition further includes an agent, which may be animmunomodulatory agent, an anti-microbial agent, an antisense molecule,or a ribozyme.

According to yet an additional aspect of the present invention there isprovided a method of identifying M. hyopneumoniae in a biologicalsample, the method comprising: (a) contacting the biological sample witha nucleic acid molecule including a polynucleotide sequence capable ofspecifically binding an M. hyopneumoniae peptide; and (b) detecting thenucleic acid molecule bound to the M. hyopneumoniae polypeptide in thebiological sample, to thereby identify the M. hyopneumoniae infection.

According to an additional aspect of the present invention there isprovided a method of treating or preventing M. hyopneumoniae infectioncomprising providing to a subject in need thereof, a therapeuticallyeffective amount of a nucleic acid molecule including a polynucleotidesequence capable of specifically binding a polypeptide participating inM. hyopneumoniae infection of cells, thereby treating or preventing theM. hyopneumoniae infection.

According to still further features in the described embodiments theproviding is implemented by: (i) administering of the nucleic acidmolecule; and/or (ii) administering a polynucleotide expressing thenucleic acid molecule.

According to still an additional aspect of the present invention thereis provided a method of targeting an anti-microbial agent to M.hyopneumoniae infected tissues, the method comprising administering to asubject in need thereof a therapeutic effective amount of theanti-microbial agent conjugated to a nucleic acid molecule including apolynucleotide sequence capable of specifically binding a M.hyopneumoniae polypeptide, thereby targeting the anti-microbial agent tothe M. hyopneumoniae infected tissue.

According to still further features in the described embodiments thepolypeptide is selected from the group consisting of P97_({768-1082}), aRNA-directed RNA polymerase core proteins (structural matrix proteins).

According to still further features in the described embodiments thepolynucleotide sequence of APT3-Cooc10 is capable of binding a region ofP97 surface protein complex defined by amino acid coordinates{768-1082}.

According to still a further aspect of the present invention there isprovided a synthetic antibody or antibody fragment comprising an antigenbinding site specifically recognizing a polypeptide including an aminoacid sequence being at least 60% homologous to P97 as determined usingthe BestFit software of the Wisconsin sequence analysis package,utilizing the Smith and Waterman algorithm, where gap creation penaltyequals 8 and gap extension penalty equals 2, wherein the polypeptidedoes not include the P97_({768-1082}) domain of M. hyopneumoniae.

According to still a further aspect of the present invention there isprovided a method of treating or preventing M. hyopneumoniae infectioncomprising providing to a subject in need thereof, a therapeuticallyeffective amount of a synthetic antibody or antibody fragment includingan antigen binding site specifically recognizing a polypeptide includingan amino acid sequence being at least 60% homologous to P97 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, wherein thepolypeptide does not include the P97_({768-1082}) domain of M.hyopneumoniae.

According to still a further aspect of the present invention there isprovided a method of identifying M. hyopneumoniae in a biological sample(i.e. diagnostic purposes), the method comprising: (a) contacting thebiological sample with a synthetic antibody or antibody fragmentincluding an antigen binding site specifically recognizing a polypeptideincluding an amino acid sequence being at least 60% homologous to P97 asdetermined using the BestFit software of the Wisconsin sequence analysispackage, utilizing the Smith and Waterman algorithm, where gap creationpenalty equals 8 and gap extension penalty equals 2, wherein thepolypeptide does not include the P97_({768-1082}) domain of M.hyopneumoniae; and (b) detecting immuno complexes including thesynthetic antibody or antibody fragment in the biological sample, tothereby identify M. hyopneumoniae in the biological sample.

According to still further features in the described embodiments thepolynucleotide is as set forth in APT3Cooc10, APT3Cooc3, APT3Cooc2,APT3Cooc4, and APT3Histo1.

According to still further features in the described embodiments thenucleic acid sequence is as set forth in APT3Cooc10, APT3Cooc3,APT3Cooc2, APT3Cooc4, and APT3Histo1.

According to still further features in the described embodiments thetargeted amino acid sequence is defined by amino acid coordinates{768-1082} of the P97 protein for APT3Cooc10, APT3Cooc3, APT3Cooc2,APT3Cooc4, and APT31-Histo1.

According to still further features in the described embodiments thetargeted amino acid sequence is defined by amino acid coordinates{768-1050} of the P97 protein for APT3Cooc10, APT3Cooc3, and APT3Histo1.

According to still further features in the described embodiments thetargeted amino acid sequence is defined by amino acid coordinates{768-925} of the P97 protein for APT3-Cooc3, APT3Cooc3, APT3Cooc2,APT3Cooc4, and APT3Histo1.

According to still further features in the described embodiments thedetecting the immuno complexes is implemented by quantifying intensityof the label following (b).

According to still a further aspect of the present invention there isprovided a nucleic acid molecule as set forth in APT3Cooc10, APT3Cooc4,APT3-Cooc3, APT3Cooc2, and APT3Histo1.

According to an additional aspect of the present invention there isprovided a nucleic acid molecule comprising at least one polynucleotidesequence capable of specifically binding a peptidic complex, wherein thepeptidic complex comprises a polypeptide expressed by M. hyopneumoniaeand involved in binding to a host organism or a host molecule expressedby the host organism which is a target of M. hyopneumoniae for treatingor preventing M. hyopneumoniae infection in a subject in need thereof.

According to an additional aspect of the present invention there isprovided a nucleic acid molecule comprising at least one polynucleotidesequence capable of specifically binding a peptidic complex, wherein thepeptidic complex comprises a polypeptide expressed be M. hyopneumoniaeand involved in binding to a host organism or a host molecule expressedby the host organism which is a target of M. hyopneumoniae for themanufacture of a medicament for treating or preventing M. hyopneumoniaeinfection in a subject in need thereof.

According to an additional aspect of the present invention there isprovided an antiviral agent for treating or preventing M. hyopneumoniaeinfection in a subject in need thereof. According to an additionalaspect of the present invention, the subject is a pig.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of theembodiments of the present invention only, and are presented in thecause of providing what is believed to be the most useful and readilyunderstood description of the principles and conceptual aspects of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for a fundamentalunderstanding of the invention, the description taken with the drawingsmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

FIG. 1 is a schematic illustration of the aptamer selection strategy.

FIG. 2 is a schematic drawing showing parallel assessment of an enrichedaptamer library.

FIG. 3 is a gel electrophoresis showing typical results from the PAGEpurification process of bound libraries.

FIGS. 4 a-j show schematic illustrations of proposed secondarystructures as generated by QuickFold 3.0 software (O M. Zuker, MFold webserver for nucleic acid folding and hybridization prediction. NucleicAcid Research, 2003, vol. 31, pp. 3406-34-15. Available at thealbany.edu website) of the APT3Cooc10 aptamer (FIG. 4 a ), the APT3Cooc2aptamer (FIG. 4 b ), the APT3Cooc3 (FIG. 4 c ), the APT3Cooc4 (FIG. 4 d), APT3Histo1 (FIG. 4 e ), the APT3Histo2 (FIG. 4 f ), the APT3Histo4(FIG. 4 g ), the APT3Histo5 (FIG. 4 h ), the APT3Histo9 (FIG. 4 i ) andthe APT3Cooc7 (FIG. 4 j ).

FIGS. 5 a-c are schematic illustrations depicting nucleic acidmodifications. FIG. 5 a shows 2′-deoxyuridines and uridines modified atposition 5. FIG. 5 b shows 2′-deoxyadenines, adenines and guanosinesmodified at position 8. FIG. 5 c shows 2′-modified uridines.

FIG. 6 is a histogram depicting binding levels of M. hyopneumoniaespecific aptamers generated according to the teachings of the presentinvention (APT3Cooc10, APT3Histo1, and APT3Cooc3) and control singlestranded aptamer to an intact M. hyopneumoniae as determined by ELISA.

FIG. 7 , is a graph showing the effects of Mycoplasma concentration onthe binding to the cilia-coated plates and on the gelatin-coated plates.

FIG. 8 is a graph showing dose response curve showing the effect ofAPT3Cooc10 aptamer of the present invention on viability of M.hyopneumoniae treated ciliated pulmonary cells as determined using thebinding cilia assays.

DESCRIPTION

The present invention relates to nucleic acid molecules,polynucleotides, synthetic antibodies, and pharmaceutical compositionsfor detecting and treating M. hyopneumoniae infection in swine.

Structure of M. hyopneumoniae

There are two surface proteins of M. hyopneumoniae that are directlyinvolved with binding onto the cilia of the epithelium cells; these arethe P97 and P102 surface proteins^([2]). P97 and P102 are encoded byMhp183 and Mhp182, respectively that are located in the same operon.There are four other surface proteins of M. hyopneumoniae, namely P116,P146, P216, and P159 that act as paralogs of P97 and P102, and havingproven “binding capabilities” to the epithelium cilia^([9-13]). WhileP97 is directly involved in the adherence of M. hyopneumoniae to thehost respiratory cilia, it is not exclusively responsible for adherenceof M. hyopneumoniae, as binding to cilia is still observed whenadherence via P97 is blocked^([14]). This observation indicates that M.hyopneumoniae utilises at least two different strategies for bindingonto the epithelium cilia; one strategy involving the direct bindingonto the cilia, and one involving the indirect binding via the formationof extra cellular matrix (ECM) complexes between the cilia and themembrane of Mycoplasma hyopneumoniae.

All the surface proteins of M. hyopneumoniae involved in binding haveonly one “recognized” transmembrane region (for each type of protein).The current literature shows that binding takes place after the cleavageof the primary adhesins (P97 and/or P102) or after the cleavage of theparalogs surface proteins (e.g. P216)^([13]). In almost every case,there is one primary cleavage site for P97, and two or three cleavagedomains for P102 and their paralogs. This implies the formation of twomain segments for P97 (e.g. F1_(P97)/F2_(P97) for P97 and P60₃₈₄/P50₃₈₄for Mhp384) or three-to-five segments (e.g. P45₆₈₃, P48₆₈₃ and P50₆₈₃ onMhp683). Since trypsin digestion and surface biotinylation experimentsshow that all two or three segments (e.g. F1_(P97) and F2_(P97) for P97,and P45₆₈₃, P48₆₈₃ and P50₆₈₃ on Mhp683) reside on the surface of M.hyopneumoniae, this proves that in the absence of membrane spanningdomains, the fragments must bind to other surface-localized componentsof either Mycoplasma or on the cilia. In all cases, the threerecombinant proteins (often referred to as F1, F2, and F3) that arespanning the surface protein do bind heparin and porcinecilia^([11-12]). This indicates that all three segments play a role infacilitating colonization of the respiratory tract of swine; either viathe direct bonding onto the cilia or via the formation of ECMs betweencilia and Mycoplasma. In other words, it is the newly created freesegments (with their N-terminal remaining anchored to the membrane) thatbind to the epithelium lung cilia or trigger the formation of complexECMs between the cilia and M. hyopneumoniae. Without cleavage of thesurface proteins, there is no binding, and thus no infection. Thepre-requisite for triggering the sequence of events leading to infectionis the presence of the right endoprotease (i.e. enzymes) to cleave theP97 and P102 or, alternatively, the cleavage of P116, P146, and P159surface proteins.

With the non-virulent J strain, after the primary cleavage of P97, thereare two segments named F1_(J97) and F2_(J97). These two cilium adhesinfragments contain ˜653 amino acids on the N-terminal side, and ˜301amino acids on the C-terminal side. F2_(J97) contains two repeat regionsR1 and R2. Both segments bind heparin. When the C-terminal F2_(J97) wassplit/cleaved into F3_(J97) and F4_(J97), such that F3_(J97) containsonly R1 and F4_(J97) contains only R2, neither of these two sub-segmentshave any affinity for heparin. The fact that both of the cilium adhesinrepeat regions (R1 and R2) are required for heparin binding suggeststhat this interaction is conformation dependent rather than due to thepresence of a specific linear amino acid motif^([15]).

From a practical point of view, the mimicking of the events that arehappening within the pulmonary system at the onset of the infectionprocess implies:

-   -   (i) the proteolytic processing (i.e. the cleavage) of the P97        surface proteins of M. hyopneumoniae, and    -   (ii) the binding of the “pre-processed” bacteria to the cilia of        the pulmonary epithelium cells (see FIG. 1A and FIG. 2 of        Jenkins et al.^([15])).

P97 and P102 proteins appear to be implicated in the infection.According to one embodiment, a therapeutic should prevent the adherenceof the P97 and the P102 onto the epithelium cells. In other words, thetherapeutic must neutralize both the P97 and the P102 binding sequencesand their paralogs. To that end, two therapeutics can be used, one toneutralize the P97 and its paralogs, and one for P102 and its paralogs.As such the therapeutic medication is a combination of two neutralizingagents. According to another embodiment, a compound fordiagnostic/detection binds to a recognizes the P97 and/or its paralogs.

P97 and its paralogs (Mh483, Mhp271, Mhp107), and Mhp683 (a paralog ofP102) bind cilia and heparin. Also, GAGs sensitive to heparinase arefound at the surface of porcine respiratory “cilia”. From theseobservations it can be inferred the following hypothesis that (i) P97binds directly to the cilia, and (ii) the paralogs of P97, as well asP102 and all its paralogs bind cilia during the early stages ofcolonization by targeting proteoglycans decorated with highly sulphatedheparin-like GAGs. In other words, there are two primary “redundant”mechanisms to insure that binding onto the cilia of the pulmonaryepithelium takes place. One pertains to direct and fast attachment tothe cilia, while the second mechanism involves the attachment viabinding to extra cellular matrix (ECM) components of the hosts (here the“hosts” are the cilia not the epithelium cell membrane per se). Bybinding to ECM proteins such as fibronectin, plasminogen t, vitronectin,collagen, etc, the pathogenic microbes are able to mediate interactionswith the host cells and, in many cases, increase their invasivecapabilities. So far, the published data shows that there are twosub-mechanisms for binding via ECM complex; one triggered by a specificprotease that cleaves the proteins at the S/T-X-F↓X-D/E sites, andanother mechanism that involves another protease and another type ofcleavage site (i e. L-N-V↓A-V-S or ATNT↓NTNTGFS).

Note that the complete P97 surface protein amino acids sequence (SEQ IDNO:11) is^([17]):

⁰⁰⁰¹MSKKSKTFKIGLTAGIVGLGVFGLTVGLSSLAKYRSESPRKIANDFAAKVSTLAFSPYAFETDSDYKIVKRWLVDSNNNIRNKEKVIDSFSFFTKNGDQLEKINFQDPEYTKAKITFEILEIIPDDVNQNFKVKFQALQKLHNGDIAKSDIYEQTVAFAKQSNLLVAEFNFSLKKITEKLNQQIENLSTKITNFADEKTSSQKDPSTLRAIDFQYDLNTARNPEDLDIKLANYFPVLKNLINRLNNAPENKLPNNLGNIFEFSFAKDSSTNQYVSIQNQIPSLFLKADLSQSAREILASPDEVQPVINILRLMKDNSSYFLNFEDFVNNLTLKNMQKEDLNAKGQNLSAYEFLADIKSGFFPGDKRSSHTKAEISNLLNKKENIYDFGKYNGKFNDRLNSPNLEYSLDAASASLDKKDKSIVLIPYRLEIKDKFFADDLYPDTKDNILVKEGILKLTGFKKGSKIDLPNINQQIFKTEYLPFFEKGKEEQAKLDYGNILNPYNTQLAKVEVEALFKGNKNQEIYQALDGNYAYEFGAFKSVLNSWTGKIQHPEKADIQRFTFHLEQVKIGSNSVLNQPQTTKEQVISSLKSNNFFKNGHQVASYFQDLLTKDKLTILETLYDLAKKQGLETNRAQFPKGVFQYTKDIFAEAEADKLKFLELKKKDPYNQIKEIHQLSFNILARNDVIKSDGFYGVLLLPQSVKTELEGKNEAQIFEALKKYSLIENSAFKTTILDKNLLEGTDFKTFGDFLAFFLKAAQFNNFAPWAKLDDNLQYSFEAIKKGETTKEGKREEVDKKVKELDNKIKGILPQPPAKPEAAKPVAAKPETTKPVAAKPEAAKPEAAKPVAAKPEAAKPVAAKPEAAKPVAAKEAAKPVAAKPEAAKPVATNTGFSLTNKPKEDYFPMAFSYKLEYTDENKLSLKTPEINVFLELVHQSEYEEQEIIKELDKTVLNLQYQFQEVKVTSDQYQKLSHPMMTEGSSNQGKKSEGTPNGKKAEGAPNQGKKAEGTPQGKKAEGAPSQQSPTTELTNYLPDLGKKIDEIIKKQGKNWKTEVELIEDNIAGDAKLLYFILRDDSKSGDPKKSSLKVKITVKQSN NNQEPESK¹¹⁰⁸

This sequence includes both the endo- and the ecto-domains of theprotein.

For therapeutic purposes the amino acid sequence for the F2_(P97)segment of Mycoplasma hyopneumoniae 232A (SEQ ID NO:12) is:

⁷⁶⁸K L D D N L Q Y S F E A I K K G ⁷⁸⁴E T T K E GK R E E V D K K V K E L D N K I K G I L P Q P P |A A K P E | A A K P V | A A K P E | T T K P V | AA K P E | A A K P E | A A K P V | A A K P E | A AK P V | A A K P E | A A K P V | A A K P E | A A KP V | A A K P E | A A K P V | A T N T G F S L T NK P K E D Y F P M A F S Y K L E Y T D E N K L S LK T P E I N V F L E L V H Q S E Y E E Q E I I K EL D K T V L N L Q Y Q F Q E V K V T S D Q Y Q K LS H P M M T E | G S S N Q G K K S E G T P N Q G KK A E | G A P N Q G K K A E G T P N Q G K K A E |G A P S Q Q S P T T E L T N Y L P D L G K K I D EI I K K Q¹⁰⁵⁰ G K N W K T E V E L I E D N I A G DA K L L Y F I L R D D S K S G¹⁰⁸²Underlined are the R1 motifs; twice underlined are the R2 motifs. Notethat F2_(P97) of Mycoplasma hyopneumoniae strain J is 301 amino acidslong, while the F2_(P97) of stain 232A is 314 amino acids long. This isbecause there are only 9 repeats in strain J while there are 15 repeatsfor strain 232A.

For detection purposes, published data suggests that only a subsectionof F2_(P97) can be used, in particular, the section that contains the R1repeats sequences. Thus, the target section (SEQ ID NO: 13) for theSELEX process is:

⁷⁶⁸K L D D N L Q Y S F E A I K K G E T T K E G K RE E V D K K V K E ⁸⁰⁰L D N K I K G I L P Q P P | AA K P E | A A K P V | A A K P E | T T K P V | A AK P E | A A K P E | A A K P V | A A K P E | A A KP V | A A K P E | A A K P V | A A K P E | A A K PV | A A K P E | A A K P V | A T N T G F S L T N KP K E D Y F P M A F S Y K L E⁹¹⁴ Y T D E K L S L T⁹²⁵

The amino acids length of the target section thus varies from 113 to 157amino acids, depending on the start (in italic-underlined) and finish(in bold black double-underlined) amino acids.

To produce critical segments of various surface proteins involved (orresponsible) for binding onto cilia of pulmonary epithelium cells,detailed procedures are described^([18]). The exact proteasesresponsible for the cleavage (i.e. the endoproteases processing of P97,P102, and their paralogs) of the surface proteins still remain unknown(see F. C. Minion,^([11]) for few possible proteases). The prevailinghypothesis is that a single protease is responsible for cleavage et allthe S/T-X-F↓X-D/E sites while another protease is responsible forcleavage et all the other sites (see ^([13]), A. T. Deutscher et al., J.of Proteome Res., 2012, vol. 11, pp. 1924-1936), and a third oneinvolves in the formation of complex ECM. While the three endoproteasesthat are operating in-vivo are not known, trypsin has been used withsuccess in-vitro to induce cleavage of the P97 surfaceproteins^([13, 19]). In all previous studies of Mycoplasmahyopneumoniae, trypsin was added to cell suspensions at concentrationsof trypsin of 0, 0.3, 0.5, 1, 3, 10, 50, and 300 μg/ml and incubated at37° C. for 15 min. It is obvious that high concentrations of trypsinwill digest the whole bacteria while the intent here is to cleave thesurface protein as fast as possible (<1 min) for detection purposes.Thus, it is suggested to use ˜1 μg/ml of trypsin but for one minute ofexposure before entering the VDC. Nevertheless, the combination of[trypsin] and time of exposure at 37° C. needs to be optimized.

For detection purposes, it is also important to account for the factthat the binding of Mycoplasma hyopneumoniae onto the porcine lungepithelium cilia is temperature dependent. This conclusion applies toboth the direct binding of P97 to the cilia and to the indirect-bindingvia the ECM-GAGs complexes. Hence binding is maximal at 37° C., weak at25° C. and minimal or inexistent at 4° C.^([20]). This stems from thefact that the biochemical reactions (i.e. the endoproteolytic processes)that are needed to cleave the various surface proteins are alsoT-dependent. This implies that Mycoplasma hyopneumoniae can survive forlong periods of time at low temperature (T≤20° C.), with the “uncleaved”surface proteins acting as camouflage (they mimic collagen and othertypical lung tissue-forming molecules). When inhaled and exposed to theoptimal temperature (i.e. T 37° C.), cleavage of the surface proteins(e.g. P97) takes place, follow by binding to cilia. From thepoint-of-view of detection, this implies that the “pre-activation” ofthe surface proteins (i.e. the cleavage of P97, P102, and their paralogswith trypsin) may be carried-out at 37° C. to increase the probabilityof detection.

As an alternative to the F2_(p97) segment being 114 amino acids long,there may be few repeated amino acid sequences that pertain to thesplitting of the P97, P102, and P146 surface proteins. Althoughdifferent, there are similarities between these cleavage motifs. Infact, the identified cleavage motifs may have similar features such as“F or V” amino acid at the “right place”, as well as first and secondneighbors that lead to similar polarity (i.e. Isoelectric point,3d-configuration, etc.). This stems from the fact that SEQ ID NO: 15includes within it a short sequence (TTKF↓QE) at amino acid residues 164to 169 that defines proteolytic cleavage in these surface proteins whichcontrol the binding of myco on the pilis of the pulmonary epithelium.For P97 there is a coiled-coil domain directly associated with thecleavage site at 195. If all the cleavage motifs have similar features(a given sequence preceded by a coiled-coil domain), they can thus berecognized by the available endoproteases (the endoproteases are neededto make the cleavage). This results in P50 (or F1, F2) segments that arevery efficient in binding to cilia and heparan sulfate. It thus appearsthat the coiled-coil+short sequence associated with cleavage is thespecific target for aptamer determination. In that case the aptamersneed to bind to the cleavage sites before the cleavage process starts;thus preventing the formation of the P50 or F1, F2, etc. segments thatinitiate direct binding and the formation of ecm-complex. The target forthis case is

L V A E F N F S L K K I T E  K L N Q Q I E N L S T K  I T N F ↓ A D E K T S S Q K S P S T L(SEQ ID NO: 14). The underlined segment corresponds to the coiled-coildomain while the twice underlined domain defines the cleavage site.

Recalling that pneumonia due to direct viral infection or due tosecondary bacterial or viral invasion is the most frequentcomplication^([21]), and given the impact of M. hyopneumoniae on theporcine industry, the challenge at present is to detect airborne M.hyopneumoniae or to generate highly potent prophylactic tools that canbe used to prevent M. hyopneumoniae infection in subjects that are atconsiderable risk of infection such as pregnant sows and young piglets.Unfortunately, measures to regulate the disease have been complicated bythe pattern of persistent, subclinical infection with occasionalepidemic outbreaks as well as the high heterogeneity of the virus andthe failure of the antibody response to completely protect againstre-infection and re-emergence^([2-3]).

The terms “sequence identity” or “% identity” in the context of two ormore nucleic acid or protein sequences, refer to two or more sequencesor subsequences that are the same or have a specified percentage ofamino acid residues or nucleotides that are the same, when compared andaligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms or by visual inspection. Forsequence comparison, typically one sequence acts as a reference sequenceto which test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are input into a computer,subsequence coordinates are designated if necessary, and sequencealgorithm program parameters are designated. The sequence comparisonalgorithm then calculates the percent sequence identity for the testsequence(s) relative to the reference sequence, based on the designatedprogram parameters. Optimal alignment of sequences for comparison can beconducted. e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection (see generally, Ausubel, F. M. et al., Current Protocols inMolecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al., J Mol.Biol. 215: 403-410 (1990) and Altschul et al., Nucleic Acids Res., 15:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(hereinafter “NCBI”). The default parameters used in determiningsequence identity using the software available from NCBI, e.g., BLASTN(for nucleotide sequences) and BLASTP (for amino acid sequences) aredescribed in McGinnis et al., Nucleic Acids Res., 32: W20-W25 (2004).

Detecting/Diagnosing Mycoplasma hyopneumoniae

The control of infectious disease is critically dependent on theavailability of appropriate diagnostic tools. Several diagnosticmethodologies are used to monitor Mycoplasma hyopneumoniae infection.These include (i) following clinical signs and abattoir surveillance,(ii) the used of bacteriological cultures, (iii) serological detectionsuch as ELISA, (iv) detection of M. hyopneumoniae antigen, (v) In situhybridisation, (vii) and Polymerase Chain Reaction (PCR) methods^([22]).As of today, the isolation of M. hyopneumoniae from affected lungs bybacteriological culture is considered the “gold standard” diagnostictechnique but isolation of the pathogen requires specialised Friismedium. When the detection of M. hyopneumoniae by culture is compared tothe detection via immunofluorescence assay (IFA) or Enzyme-LinkedImmuno-Sorbent Assay (ELISA), or by a polymerase chain reaction (PCR)method, bacteriological culture method is found the most sensitivetechnique particularly at the later stages of PEP when fewer Mycoplasmaorganisms are present^([23]). However, because the culture of M.hyopneumoniae is laborious and time-consuming (isolation from fieldsamples requires 4 to 8 weeks), several PCR techniques for M.hyopneumoniae DNA detection in different sample types have beendeveloped (see table 2 in M. Sibila et al^([22])). These PCR methods aremore rapid than bacteriological culture and are relatively inexpensiveto perform. However the confounding significance of sample contaminationis much higher with PCR. Given that M. hyopneumoniae DNA from both liveand dead organisms is amplified, the identification of PCR positiveanimals raises the question of whether such pigs have active infectionor not.

Because M. hyopneumoniae attaches to the ciliated epithelium of theairways, the best samples to detect M. hyopneumoniae by PCR aretracheobronchial swabs or the bronchoalveolar lavage fluid (BALF).Interestingly, the use of PCR to detect M. hyopneumoniae in lung tissuehas produced variable results. Hence, for moderate to severe cases ofPEP, the use of lung samples was found more appropriate than the use ofBALF^([24]), while providing misleading information for mild cases ofPEP^([25]).

Ideally, a test to detect the presence of a pathogen in a living animalshould be easy to perform, rapid, inexpensive and should provide data ofuse in the implementation of control measures. Although the detection ofM. hyopneumoniae in the nasal cavities of living pigs by PCR mighttheoretically fit these criteria pigs inoculated with M. hyopneumoniaeintratracheally were found to have low numbers of organisms in theirupper respiratory tract and only shed the organismintermittently^([22-26]). However, the use of PCR to diagnose naturalinfection from nasal swabs was found reliable and an association wasfound between the detection of M. hyopneumoniae in the nasal cavitiesand bronchi with lesions of PEP. Although the potential use of nasalswabs for nested PCR (nPCR) testing for M. hyopneumoniae in live pigshas been demonstrated^([25-26]) the procedure is currently consideredmore useful for the monitoring of infection at a herd rather than at anindividual animal level.

As an alternative to the current methodologies to detect viral andbacterial pathogens, the use of aptamers has been extensivelyimplemented in last ten years. This is clearly reflected in thepublication statistics. Since their discovery in 1990, there have been˜11,000 publications indexed to DNA or RNA aptamers in SciFinder, andmore than 1,600 of these publications are patents^([27]). Aptamers arespecific oligonucleotides composed of single stranded DNA (ssDNA) or RNAthat bind to a wide range of targets specifically. Aptamers can beobtained using an in vitro selection procedure called SystematicEvolution of Ligands by EXponential enrichment (SELEX), that starts withthe incubation of random oligonucleotide libraries with the desiredtarget molecules, followed by the separation and amplification of boundoligonucleotides^([28]). By repeating this process, an enriched pool isobtained, which can be used as a starting library for the next round ofselection to attain high specificity and affinity to the targetmolecules. FIG. 1 illustrates the continuous enrichment and selection ofthe binding aptamers to a given (specific) peptide sequence.

The production of aptamers is not costly, and they are very low inbatch-to-batch variation compared to the antibodies produced in vivo. Inaddition, aptamers can be chemically synthesized, are thermally stable,and are suitable for long-term storage^([29]). With these advantages,aptamers with high specificity and affinity have been developed for avariety of targets, including proteins, small molecules, whole cells,and viruses. Aptamers have now been widely used in diverse fields, suchas diagnostics^([30]), therapeutics^([31]), and biosensors^([32]) as analternative to antibodies.

Development of Therapeutics for Mycoplasma hyopneumoniae

Several approaches have been undertaken to uncover novel therapeuticsand vaccines against M. hyopneumoniae ^([1, 4-5, 8-9, 33]). Now, thereare few killed-virus and modified-live vaccines (MLV) are onmarket^([34]). Killed-virus vaccines are generally of limited efficacyat best^([1, 35]) while modified live M. hyopneumoniae vaccines (MLV)are the most effective option currently available for the control of thedisease. Modified live M. hyopneumoniae vaccines can confer solidprotection against homologous reinfection and have significant effectsin reducing viral shedding^([1, 8]). But the vaccine efficacy variesupon heterologous challenge. None of the current vaccines is able tocompletely prevent respiratory infection, transplacental transmission,as well as pig-to-pig transmission of the virus^([1, 8, 33, 35-36]).Furthermore, the fact that the number of seropositive animals graduallyincreases towards the end of the fattening period in both vaccinated andnon-vaccinated herds suggests that antibodies induced by either naturalinfection or vaccination do not prevent furtherinfection^([1, 4-5, 8-9, 35-36]).

Since M. hyopneumoniae is an immunosuppressive disease, i.e. it confusesthe immune defence system in the lung to stop it destroying them, ittakes up to 4 weeks for the lesions to develop; they then consolidatefor a further 4 weeks and then progressively heal from 10 weeks, as thepig's own immunity/resistance develops^([9, 23, 37]) (normally arespiratory infection such as the common cold, is cleared within 14days). This immuno-suppression allows the secondary bacteria and otherprimary bacteria such as Actinobacillus pleuropneumoniae, to gain easieraccess to the lung and, accompanied by the virus damage also to theimmune defences, can lead to the overwhelming infections.

Control of Mycoplasma hyopneumoniae Infections

Optimizing management and housing conditions is primordial in thecontrol of M. hyopneumoniae infections and should be the first to beaccomplished. Instituting management changes that reduce thepossibilities of spreading M. hyopneumoniae or result in decreased lungdamage by other pathogens do significantly improve the control ofporcine enzootic pneumonia (PEP). Additional factors different fromhousing and management conditions, such as strain differences, maydetermine the infection pattern and clinical course of the disease.Overviews of control measures for M. hyopneumoniae infections related toenvironmental and management factors have been published by Maes etal.^([13, 36]).

To control and treat respiratory disease including M. hyopneumoniaeinfections in pigs, tetracyclines and macrolides are most frequentlyused. Also, other potentially active antimicrobials against M.hyopneumoniae include lincosamides, pleuromutilins, fluoroquinolones,florfenicol, aminoglycosides and aminocyclitols. Fluoroquinolones andaminoglycosides have mycoplasmacidal effects. Since the organism lacks acell wall, it is insensitive to β-lactamic antibiotics such aspenicillins and cephalosporins. Antimicrobial resistance of M.hyopneumoniae has been reported to tetracyclines, macrolides,lincosamides, and fluoroquinoles^([7, 22, 40, 37]). This does not seemto constitute a major problem for treatment of M. hyopneumoniaeinfections to date^([1, 22, 33]).

While for most antimicrobials tested, the performance parameters areimproved and lung lesions as well as clinical signs are decreased intreated animals, the treatment and control of enzootic pneumoniaoutbreaks is disappointing because the symptoms often reappear aftercessation of the therapy. Pulse medication in which medication isprovided intermittently during critical production stages of the pigs,can also be used. However, pulse medication during extended periods oftime as well as continuous medication during one or more productionstages is discouraged because of both the increased risk of spread ofantimicrobial resistance and the possible risk for antimicrobialresidues in the pig carcasses at slaughter^([1, 22, 33]).

Commercial vaccines, consisting of inactivated, adjuvanted whole-cellpreparations, are widely applied worldwide^([34]). The major advantagesof vaccination include improvement of daily weight gain, feed conversionratio and sometimes mortality rate. Additionally, shorter time to reachslaughter weight, reduced clinical signs, lung lesions, and lowertreatment costs are observed^([9, 22, 33]). Although protection againstclinical pneumonia is often incomplete and vaccines do not preventcolonization, some studies indicate that the currently used vaccines mayreduce the number of organisms in the respiratorytract^([1, 22, 33, 38]) and may decrease the infection level in aherd^([22, 39]). On the other hand, transmission studies underexperimental and field conditions^([22, 39]) showed that vaccinationagainst M. hyopneumoniae with the current commercial vaccines inducedonly a limited and non-significant reduction in the spread of M.hyopneumoniae. Consequently, vaccination alone with the current vaccinesis not sufficient to eliminate M. hyopneumoniae from infected pig herds.In the following sections we describe the development of novelanti-microbial therapeutics.

Preparation of Target for the SELEX Process

The nucleic acid molecules of the present disclosure are selected tospecifically bind to molecules which participate in M. hyopneumoniaeinfection of a host organism. In some embodiments, the molecules are M.hyopneumoniae derived polypeptides. These M. hyopneumoniae derivedpolypeptides may be various surface proteins or fragments thereofexpressed by M. hyopneumoniae and which are used by M. hyopneumoniae tobind to the host organism's epithelium cilia. In other embodiments, themolecules are those expressed by the host organism which is the targetof the M. hyopneumoniae.

In one aspect, the nucleic acid molecules of the present disclosure aregenerated to specifically bind to a polypeptidic complex comprisingsurface proteins expressed by M. hyopneumoniae and/or molecules arethose expressed by the host organism which is the target M.hyopneumoniae. To produce the “targeted binding site” that is needed forproducing an aptamer by SELEX specific for a P97 surface protein, wefirst proceed with synthesizing the {768-1082} and the {768-925} segmentof the P97 surface protein^([17]).

Hence, the optimal amino acids “binding sequence” of P97 protein (theecto domain, SEQ ID NO:12) is:

⁷⁶⁸KLDDNLQYSFEAIKKGETTKEGKREEVDKKVKELDNKIKGILPQPP|AAKPE| AAKPV| AAKPE| TTKPV| AAKPE| AAKPE| AAKPV|AAKPE| AAKPV| AAKPE| AAKV| AAKPE| AAKPV| AAKPE| AAKPV| ATNTGFSLTNKPKEDYFPMAFSYKLEYTDENKLSLKTPEINVFLELVHQSEYEEQEIIKELDKTVLNLQYQFQEVKVTSDQYQKLSHPMMTE| GSSNQGKKSEGTPNQGKKAE| GAPNQGKKAEGTPNQGKKAE| GAPSQQSPTTELTNYLPDLGKKIDEIIKKQGKNWKTEVELIEDNIAGDAKLLYFILR DDSKSG¹⁰⁸²while the minimal domain (“binding sequence”, SEQ ID NO:13) fordiagnostic purposes is

⁷⁶⁸KLDDNLQYSFEAIKKGETTKEGKREEVDKKVKELDNKIKGILPQPP|AAKPE| AAKPV| AAKPE| TTKPV| AAKPE| AAKPE| AAKPV|AAKPE| AAKPV| AAKPE| AAKPV| AAKPE| AAKP| AAKPE| AAKPV| ATNTGFSLTNKPKEDYFPMAFSYKLEYTDENKLSLKT⁹²⁵

Various technologies are currently available to produce these twopeptides. The procedure that was used to produce P97 and N97 isdescribed below.

First, the DNA sequence encoding 315 amino acid (aa) sequence of P97 wassynthesized and cloned into a regular cloning vector. The DNA sequenceencoding MHHHHHH (SEQ ID NO: 20) was added before K768 at the 5′. Thesequence had 322×3=966 bp ORF plus two STOP codons at the end plus 5′and 3′ cloning sites (total=984 bp), and was confirmed by sequencing.The ˜984 bp fragment was cloned into an E-coli expression vector. TheN-terminal 157 aa (plus MHHHHHH (SEO ID NO: 20) plus STOPs plus cloningsites) was PCR amplified and cloned into E. coli expression vector toobtain the second expression plasmid. Both plasmids were confirmed bysequencing (the sequence has many repeats and this made it difficult tosynthesize and make expression plasmids).

Then the expression was optimized by introducing into E. coli and bytrying many different conditions.

Since the proteins were partially insoluble, they were purified underdenaturing conditions (1 L culture each) and were refolded.

The following genes were cloned into pET expression vectors. Thesequence was confirmed by sequencing the genes, and was introduced intothe E. coli expression host BL21. We analyzed several colonies forexpression at 18° C. and 37° C. by SDS-PAGE and Western with anti-Hisantibody. The results are summarized below.

-   -   Approximately 40% of the His-P97 protein (323aa (His tag and        316aa)=35.47 kDa, pI 8.56) is soluble at 18° C. with high        expression levels.    -   The His-NP97 protein (165aa (His tag and N-terminal 158aa)=17.82        kDa, pI 9.76) is soluble at 18° C. with high expression levels.    -   The His-R97 protein (His-R97, 323aa (His-tag and randomized P97        control)=35.44 kDa, pI 8.50) is soluble both at 18° C. and        37° C. but with very low expression levels.    -   The His-RBP protein (257aa (His-tag and mouse RBP control)=26.40        kDa, pI 9.95) showed no expression both at 18° C. and 37° C.    -   The His-GFP protein (245aa (His-tag and GFP control)=27.76 kDa,        pI 5.99) is soluble both at 18° C. and 37° C. with high        expression levels.    -   Approximately 50% of the experimental control GST-His protein        (256 aa=29.79 kDa, pI 7.19) is soluble at 18° with high        expression levels.

Afterward, large scaled cultures of His-P97, His-NP97, His-GFP, andHis-RP97 were grown and purified.

Determination of the ssDNA Aptamers for M. hyopneumoniae

Using the P97_({768-1082}) and the P97_({768-925)} peptides, thecorresponding binding aptamers were identified using the SELEXprocedure. FIG. 1 shows the overall procedures that were used toidentify the aptamers for the targeted peptides.

Electrophoretic Mobility Shift Assays (EMSAs) were used to separateportions of the aptamer library that bind to a given target (NP97) andfail to bind to a counter target (RP97) in a buffer consisting of 1×DPBS(26.67 mM KCl, 14.71 mM KH₂PO₄, 1.38 M NaCl, and 80.60 mM Na₂HPO₄-7H₂O)with 10 mM MgCl₂ (pH 7.4). One round of selection consisted of enrichingfor unbound, non-shifted DNA material in the presence of the countertarget followed by the isolation of bound and shifted DNA after exposureto the target molecule. Each selection round was followed by libraryamplification through PCR and purification of the DNA Sense strand Aftersubjecting the initial library of diverse random sequences to threeconsecutive rounds of selection, the enriched library was divided intotwo fractions to perform the parallel assessment as shown in FIG. 2 .

Aptamers enriched after three rounds of positive and negative selectionsare subjected to a parallel assessment, which involves simultaneouslyexposing half of the enriched library of aptamer to the target and theother half of the library to the counter target. The parallel assessmentis designed to identify DNA molecules that bind indiscriminately to bothtarget and counter target molecules. These promiscuous aptamers werediscarded during the bioinformatics analysis portion of the project.

After parallel assessment PAGE purifications of bound libraries wereperformed, 15 pmoles of enriched library was exposed separately toeither the target (NP97) or counter target (RP97) in selection buffer.After a 60-minute incubation at 37° C., bound DNA material was separatedfrom unbound material using 10% non-denaturing PAGE. Presumably boundmaterial was excised, eluted from the gel, and used as template in PCRamplification in preparation for sequencing. Gel images were takenbefore and after excision. Hyperladder V (Bioline; Randolph, Mass.) wasused as a molecular weight standard. The gels were stained with GelStarNucleic Acid Stain (Lonza; Walkersville, Md.). Typical results are shownin FIG. 3 .

Predicted secondary structures (top panels) and sequence data (bottompanels) are displayed in FIG. 4 for the five of the ten most promisingaptamer candidates. The first four candidates (FIGS. 4 a to 4 d ) andthe last candidate (FIG. 4 j ) were determined using motif analysis,while the remaining five (FIGS. 4 e to 4 i ) were determined on thebasis on sequence frequency in the candidate library dataset. Thesecondary structure and free energy for each aptamer was computed withQuickFold 3.0 (O M. Zuker, MFold web server for nucleic acid folding andhybridization prediction. Nucleic Acid Research, 2003, vol. 31, pp.3406-34-15. Available at the albany.edu website). Sequences displayedbelow each structure were identified using proprietary algorithms ofAptagen. The algorithms are improved versions of the COMPAS algorithm.COMPAS can be downloaded from the aptait.de website. The underlinedregion in each sequence is the PCR prime annealing region.

The initial library containing a large number of random sequences wassubjected to three rounds of polyacrylamide gel-based SELEX. The SELEXprocess is designed to enrich for sequences that bind to the targetmolecule and eliminate sequences that bind to the counter targetmolecule over multiple rounds of selection (FIG. 1 ). As a result, thepopulation to be sequenced is expected to contain multiple copies ofpotential aptamer candidates; largely homologous sequences representinga library of aptamer candidates^([41]). The selection strategy employedin the present project was designed to identify aptamers that bind toNP97, what has been determine to be a region of protein vital for thepathogenicity of M. hyopneumoniae, but fail to bind to RP97, arandomized version of that same region. Three rounds of positive andnegative selections were conducted. The resultant enriched library wasPCR amplified and then divided into two samples, with one exposed to thetarget in binding buffer and the other exposed to the counter-target(negative-target) in binding buffer (FIG. 2 ). After partitioningthrough non-denaturing polyacrylamide gel electrophoresis(non-denaturing PAGE), the regions of gel containing presumably boundmaterial were excised for nucleic acid elution (FIG. 3 ). DNA collectedfrom the elution was PCR amplified in preparation for sequencing.

Illumina-based technology was implemented to sequence the aptamers afterthe selections. Subsequent bioinformatics analysis of the sequencingdata identified candidate aptamer molecules. Deep sequencing andsubsequent data analysis eliminated the traditional approach ofperforming a large number of selections, which may introduce error andbias due to the screening process.

A “good sequence” was assessed as one that contained the full forwardand reverse primers (or their complements), as well as a variable regionbetween 44 and 54 nucleotides long to account for minor insertions ordeletions of bases. The data was analyzed using proprietary algorithmsto identify candidate sequences.

For the aptamer candidate selection the analysis ranked sequencesaccording to a variety of categories, including frequency of homologoussequences, frequency based on motif presence, and presence of multiplemotifs. A motif is a smaller segment of bases conserved betweensequences, most likely because the segment contributes to the aptamer'sbinding ability. For the selection of ten candidate sequences to test,the presence of multiple motifs in a sequence was weighted more heavilythan the frequency of homologous sequences. However, the relativecomplexity of a candidate's secondary structure weighed heavily in itsranking. This was primarily determined by the number of stems on thepredicted secondary structure (as described below) that are coming-outof the central junction. Thus, while APT3Cooc7 (FIG. 4 j ) wasdetermined through the co-occurrence method^([41]), the fact that thereis only 1 stem coming from the central junction makes it one of the lesslikely candidates. Despite this, there is no large difference in therankings of the candidates, and they are all worth examining for furthercharacterization.

In addition to sequence and motif analysis, secondary structureprediction of the candidate sequences was carried out using the MfoldWeb Server (O M. Zuker, MFold web server for nucleic acid folding andhybridization prediction. Nucleic Acid Research, 2003, vol. 31, pp.3406-34-15. Available at the albany.edu website). By inputting the fullsequence as well as folding temperature and salt conditions, it ispossible to make an informed prediction about what structures an aptamercandidate may take (FIG. 4 ). The parameters used for the analysis ofthe selected sequences were matched as closely to selection parametersas possible (e.g. 1.5 M NaCl, 0.01 M MgCl₂).

The selected aptamer candidates were then synthesized usingphosphoramidite chemistry, and used for affinity binding studies andK_(d) measurements.

From FIG. 4 the ten (10) aptamers corresponding to the P97 peptidiccomplex targets of M. hyopneumoniae are:

APT3Cooc10 (SEQ ID NO: 1):5′-GTT-TGT-TGT-GAG-CCT-CCT-AAC-GGG-TAT-GAC-TAC-AGA-TGC-AGG-GCG-GCC-TGT-AGC-CTT-GCA-TTG-ACA-AGG-GCA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Cooc2 (SEQ ID NO: 3):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-GCG-AGT-CCC-AAT-CTG-GAG-GGG-AGC-GAG-AGG-CAA-GTA-TGG-TTG-CCG-GGA-GCA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Cooc3 (SEQ ID NO: 5):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-TAG-CTC-GTA-GAA-AAA-AAA-TAT-AGC-GTG-TGC-TGG-GAC-TGC-TCG-GGA-TTG-CGG-ACA-CAT-GCT-TAT-TCT-TGT-CTC-CC-3′ APT3Cooc4 (SEQ ID NO: 2) 5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC -AGT-GAT-GAA-GGG-ATC-ACG-GGC-AAA-GGA-CCG-TGA-CAA-ATC-ACG-GAG-TGT-CAT-GCT-TAT-TCT-TGT-CTC-CC -3′ APT3Histo1 (SEQ ID NO: 4):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-CGC-ACG-TGG-GTA-TTC-TAA-GTG-CGG-TAG-CTC-AAT-GGT-GAG-CGA-TGA-GCA-TCA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Histo2 (SEQ ID NO: 6):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-GAA-AGT-TAC-AGG-CTG-CGC-GGA-GAG-GAG-CCC-AAG-CGA-GCT-TTG-CTG-ATG-CCA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Histo4 (SEQ ID NO: 7):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-ATA-GCA-CAT-TTG-ATG-AGG-AGG-CCT-TGA-TTA-AAG-GCC-GGC-TTG-TGA-ACG-TCA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Histo5 (SEQ ID NO: 8):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-GAC-GAT-GGA-GCG-GCC-CCA-AGT-CGC-TCT-TGC-ATG-TTA-ATG-GAT-CGC-CAC-ATC-ATG-CTT-ATT-CTT-GTC-TCC-C-3′ APT3Histo9 (SEQ ID NO: 9):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-GTT-GCC-GCT-TAG-TGG-GCG-GCA-TCA-CTC-GAT-TGA-AAC-GAT-TAG-TGT-AGT-ACA-TGC-TTA-TTC-TTG-TCT-CCC-3′ APT3Cooc7 (SEQ ID NO: 10):5′-GCC-TGT-TGT-GAG-CCT-CCT-AAC-ACC-ACG-GGC-CTG-GGG-CAT-TTA-TAG-CAT-AGG-CGC-GTG-GCA-ACC-TAC-GTA-CCA-TGC-TTA-TTC-TTG-TCT-CCC-3′

Using the most appropriate cilia binding procedure these aptamers havebeen “screened” to identify the ones having the highest and fastestbinding capabilities for detection and therapeutics purposes. Hence, weused a tissue culture system^([42]) to study the binding of our aptamersonto the ciliary epithelial. The differentiated epithelial cell modelthat was used has distinct advantages for the study of the interactionsbetween M. hyopneumoniae, the aptamers and the tracheal epithelialcells. This is because the morphology of the cultured cells shares manyof the features found on the intact epithelium. Most prominent featureof the culture system is the presence of tight junctions and thedifferentiation of the apical and basolateral cell membranes. The apicalmembrane contained cilia and microvilli while the basolateral membraneis showing interdigitations. The presence of mitochondria, endoplasmicreticulum, secretory granules, and basal bodies of cilia are similar tothat observed in intact epithelium. The cells grown with the airinterface as described here differentiated as demonstrated by theformation of cilia. Under the culture conditions described,approximately 10 to 50% of the cells are covered by cilia in contrast tonormal swine epithelium, which has at least 70% of the surface cellsciliated^([43]).

Preparation of Cilia Model

Previous work has provided information relative to the attachment of M.hyopneumoniae to and effect on, the cilia in several different swinecell systems. These systems have included single ciliated cells fromswine tracheas^([43]) newborn piglet tracheal organ cultures^([44]) anda microtiter plate adherence assay using purified swine tracheal cilia(Zhang,^([20])). None of these systems allowed close simulation of theepithelial cell/Mycoplasma relationship that is thought to be such adynamic aspect of the infection in the living pig^([45]). This is why weused a tissue culture system consisting of confluent monolayers oftracheal epithelial cells on microporous membranes. The cells retainmost of the morphologic characteristics of intact tracheobronchialepithelium including apical microvilli and cilia, apical tightjunctions, moderately interdigitated lateral intercellular space andmucin secretion

Epithelial cells were isolated from the tracheas of twelve-weeks old SPFpigs (SIV, PRRSV, and M. hyopneumoniae-free). The pigs were euthanizedby percussion followed by exsanguination. The tracheas were asepticallyremoved and rinsed in cold sterile phosphate buffered saline solution,pH 7.2 (PBS) to remove mucus and debris. All muscle tissues were trimmedoff the tracheas, which were then placed in 50 ml polypropylene tubescontaining sterile 0.15% pronase and 0.01% DNAse in Ca²⁺- and Mg²⁺-freeminimum essential medium (MEM) and incubated at 4° C. for 24 h. Theenzymes were inactivated after 24 h with the addition of foetal bovineserum (PBS) to a final concentration of 10%.

The tubes were inverted several times to loosen cells from the tracheas,and cells were pelleted by centrifugation at 125 g for 5 min. Thepelleted cells were re-suspended in a mixture of Dulbecco's MEM (highglucose) (DMEM) and Ham's F-12 (1:1) containing 5% FBS, 0.12 U/mlinsulin, and 100 U/ml of penicillin-streptomycin. Cell suspensions weretransferred to tissue culture dishes and incubated in 5% CO₂ for atleast 1 h to remove fibroblasts. The non-attached epithelial cells werethen collected. A portion of the cell suspension was diluted in 0.04%trypan blue for counting and assessing viability.

Millicell-PCF inserts (0.45 μm pore size, 0.6 cm² area, Millipore,Bedford, Mass., USA) were coated with human placental collagen (HPC;Type IV, Sigma) and placed in 24-well culture plates. A stock solutionof 0.05% HPC in 0.2% glacial acetic acid was prepared. EachMillicell-PCF insert was coated with 200 μl of a 1:10 dilution of thestock HPC solution and incubated overnight at room temperature. On theday the tracheal cells were added to the inserts, the collagen wasremoved and the inserts air-dried. The inserts were then washed oncewith PBS and once with medium to remove any residual collagen.

The tracheal cells were plated at a concentration of 3.75-5×10⁵cells/cm² onto the prepared Millicell-PCF inserts and grown on theair-liquid interface^([20, 46]). To create an air-liquid interfacefeeding system, the apical side of the membrane containing the trachealepithelial cells was left exposed to the air. The cells were nourishedfrom underneath with serum-free DMEM/F-12 (1:1) containing 2% ultroser Gserum substitute (USG medium) supplemented with penicillin andstreptomycin.

Mucin secreted by the cell cultures was measured by a mucin dot blotassay while cell secretions were collected by washing the surface ofcultures with 200 μl of PBS. 2 μl of the cell secretions in PBS, alongwith various concentrations of mucin as controls, were pipetted ontonitrocellulose membrane sheets. The membrane sheets were soaked intris(hydroxymethyl)aminomethane (THAM)-saline-polyoxyethylenesorbitan/Tween 20 (TST) for 30 min and incubated with diluted rabbitserum produced against porcine mucin type III from porcine stomach(Sigma, St. Louis, Mo., USA) at room temperature for 1 hour. The sheetswere then reacted with goat anti-rabbit horseradish peroxidase conjugatefor 1 h at room temperature. After washing with TST, color was developedwith the addition of H₂O₂ and 4-chloro-1-naphthol. The intensity ofcolor was used to estimate the quantity of mucin. The image of the dotswas captured using a Molecular Device flatbed scanner (model Emax).Intensity of each dot on the sheet was analyzed using the NIH imageanalysis software (see http://rsb.info.nih.gov/nih-image/).

Procedure for Binding Tests

All strains of M. hyopneumoniae were prepared from stock culturesinoculated into Friis medium at a 1:10 dilution followed by incubationat 36° C. in a shaking water bath. Cells were harvested when culture pHwas lowered to approximately 6.8 after 1-2 days' incubation. Cells werewashed once in PBS or Friis medium with no antibiotics and re-suspendedat 1/20 of the original culture volume in PBS or Friis medium. Theorganisms were then passed three times through a 25-gauge needle todisperse aggregates. The M. hyopneumoniae suspensions were used for theadherence study.

50 μl (5×10⁸-1×10⁹) of M. hyopneumoniae 232 was added to duplicateinserts containing the differentiated tracheal cell cultures after 12-22days of growth. The cells were then incubated at 37° C., 5.2% CO₂, foreither 90 min or 2 days. After incubation, the inserts were gentlywashed three times with PBS to remove the unattached mycoplasmas.

Cell numbers on the inserts were determined as described by Clark etal.^([47]) Briefly, tracheal cells were released from thecollagen-coated membranes by incubation in a solution of 0.05%trypsin-0.53 mM EDTA at 37° C. for 10 min. The solution was added to thetop and bottom compartments. At the end of incubation, the trypsin-EDTAsolution was carefully removed without dislodging the cell layer. 100 μlof PBS was then added to the insert and pipetted up and down extensivelyto dislodge and disperse the cells. After removing the cell suspension,membranes were washed with another 100 μl of PBS that was later pooledwith the cell suspension. A portion of the pooled cell suspension wasthen stained by trypan blue for counting in a haemocytometer.

Note that with the procedure that was used the confluent monolayers ofpseudo-stratified epithelium containing morphologically identifiableciliated, secretory and basal cells were formed on HPC-coated Millicellmembrane inserts. The cells began to differentiate 4-5 days afterplating at a density of 3.5-5×10⁵ cells/cm² (approximately 3×10⁵ perinsert) and culturing in serum replacement medium with air-liquidinterface feeding. Also, the ciliated cells are distributed throughoutthe inserts. The majority of the cells have microvilli covering thesurface of the cultured cells. The cilia on the inserts in the cultureswere healthy after 22 days in culture. The percentage of ciliated cellson the inserts varied between 10 and 50%. The number of viableepithelial cells is in the range of 6.8×10⁵ (15 days post plating) and5×10⁵ (23 days post plating) per insert.

In PBS, M. hyopneumoniae strain 232 are attached to the cilia, inducingtangling, clumping and longitudinal splitting within 90 min of theaddition of mycoplasmas to the cells. Cilia remained damaged 2 daysafter M. hyopneumoniae infection even if no organisms are present. InFriis medium, cilia were bundled together and large numbers of M.hyopneumoniae are found predominately to adhere to the top of the ciliaat 90 min and 2 days post infection. No damage to cilia was observed ateither 90 min or 2 days post infection with the 232 strain.

In PBS and with the proper (sufficient) concentrations of the bindingaptamers, M. hyopneumoniae 232 did not attach to cilia and the ciliaremained undamaged even after 22 days. In fact, after screening is wasfound that APT3Cooc10, APT3Histo1, and APT3Cooc3 have the highestbinding capabilities from a biosensing and therapeutic perspectives.

While reducing the present invention to practice, the present inventorshave uncovered that oligonucleotides (e.g., aptamers) designed to bindconserved sequences in the P97 polypeptidic complex can be utilized toprevent binding to the cilia of the pulmonary epithelium cells. As isillustrated in the examples section that follows, the present inventors,have provided, aptamer nucleic acid molecules, which can be used todiagnose and treat M. hyopneumoniae infection. Such aptamer moleculesexhibit viral cross-reactivity and as such can be used as therapeuticsand/or vaccines against the M. hyopneumoniae.

Assessing the Binding Capabilities of the Selected Aptamers

Aptamers are nucleic acid sequences of tertiary structures, which areselected to specifically bind a polypeptide of interest and inhibit aspecified function thereof. Detailed description of aptamers andmechanism of action thereof is provided by Wilson & Szostak, andothers^([28]). Thus, according to one aspect of the present inventionthere is provided a nucleic acid molecule including a polynucleotidesequence that is capable of specifically binding a polypeptidic complexparticipating in the M. hyopneumoniae infection of cells.

The ability of the nucleic acid molecules of this aspect of the presentinvention to specifically bind a polypeptidic complex that participatesin the M. hyopneumoniae infection of cells allows the use thereof in M.hyopneumoniae infection therapy and diagnostics.

As used herein “a polypeptide which participates in M. hyopneumoniaeinfection of cells” refers to a polypeptide that is encoded by variousvirulent M. hyopneumoniae strains, a host cell polypeptide or a peptidefragment thereof.

Examples of M. hyopneumoniae polypeptides that participate in infectionof pulmonary epithelium cells include P97_({768-1082}),P97_({768-1050}), P97_({768-925}), P116_({700-1010}), P102_({1-324}),and P102_({1-529}). It is important to emphasize that all these peptidescarry the repetitive motif |AAKPE|AAKPV|AAKPE|TTKPV| (SEQ ID NO. 19) (ora motif being more than 80% similar).

Examples of host cell polypeptides that participate in M. hyopneumoniaeinfection include but are not limited to P97, P102, P116, P146, P216,and P159

It will be appreciated that polypeptide targets of this aspect of thepresent invention are preferably microbial, to maximize specificity ofthe nucleic acid molecules of the present invention and reducecytotoxicity thereof. Accordingly, preferred polypeptide targetsequences include if nucleic acid molecules generated to bind suchsequences can be used as universal M. hyopneumoniae vaccines.

Few examples of conserved microbial peptide targets for M. hyopneumoniaeare provided in the following Table 1.

TABLE 1 Microbial peptide targets of M. Hyopneumoniae <SEQ ID NO>Peptide sequence Identifier Ref. P97⁷⁶⁸KLDDNLQYSFEAIKKG ⁷⁸⁴ETTKEGKREEVDKKVKE AAB47806.1 [17] <12>LDNKIKGILPQPP| AAKPE| AAKPV| AAKPE| TTKPV| andAAKPE| AAKPE| AAKPV| AAKPE| AAKPV| AAKPE| A MHP0198AKPV| AAKPE| AAKPV| AAKPE| AAKPV| ATNTGFSLT MHP183NKPKEDYFPMAFSYKLEYTDENKLSLKTPEINVFLELVHQSEYREQEIIKRLDKTVLNLQYQFQEVKVTSDQ YQKLSHPMMTE|GSSNQGKKSEGTPNQGKKAE|GAPNQGKKAEGTPNQGKKAE| GAPSQQSPTTELTNYLPDLGKKIDEIIKKQ¹⁰⁵⁰ GKNWKTEVELIEDNIAGDAK LLYFILRDDSKSG¹⁰⁸² P102⁶⁸³EDNPEGDWITLGRMEKLVKEVIQYKKEGTKTFLD MHP0182 L. <15>DEVAKTLYYLDFHHLPQSKKDLEEYKEKHKNKFIN SeymourEKPATATSQAKPDQAKNEKEVKPESAQAESSSSNSN et al,DSNSKTTSSSSMAGTTQNKSTBETPNSSSNSTPTSSA 2012 ^([16])TTSTTSSTQAAATSASSAKVKTTKFQEQEKQQVKEQ KQKQEKTKETNQLLDTKTNKENLGGLILWDFLYNSKYKTLPGTTWDFLVEPDSFNDRLKITAILKENTSQAKSNPDSKNLTSLTRNLIIKGVMANKYIDYLVQEDPVLLVDYTRRNQIKTEREGQLIWSQLASPQMASPEPEKTKLEITEEGLRVKKGGTKIKEGIKNGSSRGNTNTNS KPNKKLVLLKGAIKNPGTKKEWILVGSGIKDNNNGGSNNNSNTQIWITRLGTSVGSLKTEGETVLGISNNNSQEVLWTTIKSKLENENPSDNNQIQYSPSTHSLTTNSRSNTQQSGRNQIKITNTQRKTTTSPSQNLSQNPDPNQIDVRLGLLVQDKKLHLWWIANDSSDEPEHITIDFA EGTFNYDDLNYVGGLLKNTTNNNNTQAQDDEGDGYLALKGLGIYEPPDDESIDQAATVEKAERLYKHFMG LFRE ¹²⁰¹ P116⁶⁵¹QLTQEGFKLTNPIKFQQNQSKTKENIARTVNISYL MHP0108 L. <16>AFKPKNINDYKKHYLLADSDGNGLFIQKIKNTEKTI SeymourQNSDITFIKPENLDQKNKDETQQKQVDGSYLYQNK et al,KSLYSLANLFPPELIDKQAVILGPNSLAIVELANRIG 2010 ^([13])ENRFYRQELRNSSPFSLEKSKESVEISAFSSSNYQLNSKTSLNLNGKTIYNINPVIGPNPKKTTDKNGSNNEKI NKNSSIILKGIAVYRNAFIKAYIK ¹⁰¹⁰ P146⁵⁰⁴ NKIGIDLGVLKKYISNNQGIEYTFDIANAKIRDAQ MHP0663 D. <17>GDGITSHIEIPVTISLWSSFFGDSDNVLLKSKTETFII and BogemaPYFQKETTSESKDQKVGHTQKELDLNQKLVYQLSE MHP684 et al.,LPGTSTQGSSGSSTQTEQIKEVKLPTLTAFISKQELE 2012^([13])ALIDGDKNLASQPTSQAVSVSQVKATEFQQQDANSTNSSPTSPSPSPTSPSPASPSSSPSPTSPKNLDENIGVPNPRFEEIKKIISSEFIYKYNFRANEALLDAWVGKQNFPSLKDISQFRSDQRLAKDYKLVNLKSNKFLEDYDVLAFYANLVQKDPREVLQYLFEIARANNLIGPKEKLDLNQIEEDGIFRRAKAIKLIDKSSNNQGIYGPSFNNQFLKFHERGWMSTLYLPNEAKTKLADYQNLLSAGISDTKIFSELNKIQPLDLNIKTQSSDSSDKSDSSDSDDAKTTSTKQDLLKLTSLKSQIEAIVKKYETESKKYLG ¹⁰²⁵ P216⁹⁰³SGAKSTIIFEEIAELDPKVKEKVGADVYQLKFHYA MHP0496 J. Wilton <18>IGFDDNAGKFNQEVIRSSSRTIYLKTSGKSKLEADTI and et al. ^([13])DQLNQAVKNAPLGLQSFYLDTERFGVFQKLATSLA MHP493VQHKQKEKTLPKKLNNDGYTLIHDKLKKPVIPQISSSPKKDWFEGKLNQNGQSQNVNVSTFGSIIESPYFST NFQEDADLDQDGQDDSRQGNNSLDNQEAGLLKKLAILLGNQFIQYYQQNDKEIEFEIINVEKVSELSFRVEFKLAKTLEDNGKTIRVLSDETMSLIVNTTIEKTPEMS AVPEVFDTKWVRQYDPRTPLAAKTKFVLKFKDQIPVDGSGNISDKWLASIPLVIHQ QMLRLSPVVKTIREL GLKTEQQQQQQQQQQQQQPQKKAVRKEFELETYNPKDEFNILNPLTKAHRLTLSNLVNNDPNYKIEDLKYIKNEAGDHQLAFSLRANNIKRLMNTPITFADYNPFF YYNEDWRSIDKYLNNKGNVSSHQQQAAGGNQGSGLIQRLNKNIKPETFTPALIALKRDNNTNLSNYSDKIIM IKPKYLVERS¹⁴⁴⁴

The nucleic acid molecules of this aspect of the present invention referto single stranded or double stranded DNA or any modifications thereof,which are capable of specifically binding the polypeptide-targetsdescribed hereinabove. The nucleic acid molecules of this aspect of thepresent invention are interchangeably referred to as “aptamers”.

Typically, the nucleic acid molecules according to this aspect of thepresent are of varying length, such as 10-100 bases. It will beappreciated, though, that short nucleic acid molecules (e.g., 10-40bases) are preferably used for economical, manufacturing and therapeuticconsiderations, such as bioavailability (i.e., resistance to degradationand increased cellular uptake).

According to embodiments of this aspect of the present invention, thenucleic acid molecules are preferably those set forth in APT3Cooc10,APT3Histo1 and APT3CooSeq3.

Modification of Nucleic Acid Sequences

As mentioned hereinabove, the nucleic acid molecules of this aspect ofthe present invention are preferably modified to obtain enhancedbioavailability and improved efficacy to the target polypeptide.Modifications include but are not limited to chemical groups thatincorporate additional charge, polarizability, hydrogen bonding,electrostatic interaction and fluxionality to the nucleic acid bases orto the entire molecule. Added or modified chemical groups are selectedto include conformationally flexible linkages, which conform to thetopology of the polypeptide target. Additionally, measures are takenthat the chemistry for the modification of the nucleic acid molecules ofthis aspect of the present invention allows for either trisphosphate(NTP) or phosphoramidite synthesis.

Thus, for example, nucleic acid molecules of this aspect of the presentinvention preferably include modifications that allow specificcross-linking to the target polypeptide to thereby form high affinitycompounds.

Appended cross-linking groups can contain hydrophobic, hydrophilic orcharged functionality. Cross-linking may be accomplished by theformation of imine, acetal, ester and disulfide linkages as well as byconjugate addition to α, β-unsaturated carbonyl linkers. Examples of3′-deoxyuridine nucleosides which are suitable for phosphoramiditesynthesis are shown in FIG. 5 a including small hydrophobic functionalgroups such as vinyl (group 1, FIG. 5 a ), large hydrophobic functionalgroups such as pyrenyl (groups 13-14, FIG. 5 a ) and carbonyl compoundswith varying degrees of side chain hydrophobicity (groups 3, 6-11, FIG.5 a ).

Pyrimidine base modifications, such as DNA uridine nucleosidemodifications at position 5, can include hydrophobic groups which can beconjugated in the form of ketones (ex. groups 17, 18 FIG. 5 a ), amides(groups, 24, 27, FIG. 5 a ) and the like, which can be attached toeither DNA or RNA nucleic acid molecules^([48]). It will be appreciatedthat amides can impart hydrogen-bonding capabilities to the aptamer. Inany case, as described hereinabove, cross-linking carbonyl groups can beattached to the 5-position of uridine (groups 15-18, FIG. 5 a ). It willbe appreciated, though, that the expected reactivity of carbonyl linkerscan differ significantly depending on the interface of the targetpolypeptide.

Examples of purin modifications are shown in FIG. 5 b . For examplehydrophobic substituents can be attached at the 8-position of DNA purinenucleosides (groups 28-30, FIG. 5 b ). The degree of steric hindrancecan be varied via amide linkages (groups 31, 33, 34, 37 and 38, FIG. 5 b). Hydrophilic (group 35, FIG. 5 b ) and charged (groups 36 and 39, FIG.5 b ) groups may be appended to the 8 position of purine nucleosides. Itwill be appreciated that functional groups with known affinity to thetarget polypeptide can be attached to the 8 position of the purine base,such as a biotinylated nucleoside (group 40, FIG. 5 b ).

Additional sites for modifications include but are not limited to the3′-position of DNA. A 3′-position pyrimidine nucleoside modification canbe implemented. Essentially, amine linkers, such as hydroxyl aminelinkers can be used to attach hydrophobic groups with differenttopologies (groups 41-43, 46 and 49, FIG. 5 c ), hydrophilic groups (45and 47, FIG. 5 c ) and groups exhibiting specific affinity to the targetpolypeptide (group 45, FIG. 5 c ).

As mentioned hereinabove, the nucleic acid molecules of this aspect ofthe present invention can also be modified to increase bioavailabilitythereof. The following illustrates non-limiting examples for suchmodifications.

The nucleic acid molecules of this aspect of the present invention maycomprise heterocyclic nucleosides consisting of purines and thepyrimidines bases, bonded in a 3′ to 5′ phosphodiester linkage.

Preferably used nucleic acid molecules are those modified in eitherbackbone, internucleoside linkages or bases, as is broadly describedhereinunder. Such modifications can oftentimes facilitateoligonucleotide uptake and resistance to intracellular conditions.

Specific examples of nucleic acid molecules useful according to thisaspect of the present invention include oligonucleotides containingmodified backbones or non-natural internucleoside linkages.Oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone, as disclosed in U.S. Pat. Nos.3,687,808; 4,469,863; 4,476,301; 5,023,243, 5,177,196, 5,188,897;5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361;5,625,050, each of which are incorporated by reference in theirentirety.

Preferred modified nucleic acid backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, amino alkyl phosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms can also be used.

Alternatively, modified nucleic acid backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; 5,677,439, each of which are incorporated by reference intheir entirety.

Other nucleic acid molecules which can be used according to the presentinvention, are those modified in both sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for complementation with theappropriate polynucleotide target. An example for such a nucleic acidsequence mimetic includes peptide nucleic acid (PNA). A PNAoligonucleotide refers to an oligonucleotide where the sugar-backbone isreplaced with an amide containing backbone, in particular anaminoethylglycine backbone. The bases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Other backbone modifications, which can be used in thepresent invention are disclosed in U.S. Pat. No. 6,303,374, which isincorporated by reference in its entirety.

Nucleic acid molecules of the present invention may also include basemodifications or substitutions. As used herein, “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).Modified bases include but are not limited to other synthetic andnatural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Further bases include those disclosed in the opened literature^([49]).Such bases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine, 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2 C.^([50]) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

Another modification of the nucleic acid molecules of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates, which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. Such moieties include but arenot limited to lipid moieties such as a cholesterol moiety, cholic acid,a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-racglycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterolmoiety^([51]).

It is not necessary for all positions in a given oligonucleotidemolecule to be uniformly modified, and in fact more than one of theaforementioned modifications may be incorporated in a single compound oreven at a single nucleoside within an oligonucleotide.

As is illustrated in the following examples, the present inventors haveconclusively shown that the nucleic acid molecules of the presentinvention are capable of preventing M. hyopneumoniae infection of cellsin vitro and in vivo. Furthermore, the ability of the nucleic acidmolecules of the present invention to inhibit viral spread followingviral challenging, suggests the use of the nucleic acid molecules of thepresent invention in anti-M. hyopneumoniae prophylactic and therapeuticapplications.

Method of Treating Infection

Thus, according to another aspect of the present invention there isprovided a method of treating M. hyopneumoniae infection.

As used herein the term “treating” refers to preventing M. hyopneumoniaeinfection or substantially reducing (i.e., alleviating or diminishing)symptoms associated with M. hyopneumoniae infection.

The method is implemented by providing to a subject in need thereof, atherapeutically effective amount of the nucleic acid molecule of thepresent invention described hereinabove.

As used herein “a subject in need thereof” refers to a subject sufferingfrom M. hyopneumoniae infection associated symptoms or at risk ofcontracting M. hyopneumoniae infection. Examples of such subjectsinclude but are not limited to piglets aged 6 weeks or less; mature pigs12 weeks or more, pigs suffering from chronic diseases such as PRRSVinfection, pregnant sows, and pigs in close or frequent contact withanyone at high risk.

Preferably, the nucleic acid molecules of the present invention areprovided at a concentration of between, 0.1-150 μg/Kg body weight,preferably 1-100 μg/Kg body weight, more preferably 1-50 μg/Kg bodyweight and even more preferably 1-15 μg/Kg body weight.

Prior to, concomitant with or following providing the nucleic acidmolecule of the present invention an agent can be provided to thesubject. An agent can be a molecule that facilitates prevention ortreatment of M. hyopneumoniae infection or clinical conditionsassociated with PRRS infection such as pneumonia. Examples of agents,according to this aspect of the present invention include, but are notlimited to, immunomodulatory agents (e.g., synthetic antibodies),antibiotics, antiviral agent (e.g., amantidine), antisense molecules,ribozymes and the like.

The Pharmaceutical Composition

The nucleic acid molecule (i.e. active ingredient) of the presentinvention can be provided to the subject per se, or as part of apharmaceutical composition where it is mixed with a pharmaceuticallyacceptable carrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the preparationaccountable for the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. The term “adjuvant” isincluded under these phrases.

Since activity of aptamers is directly correlated with a molecularweight thereof, measures are taken to conjugate the nucleic acidmolecules of the present invention to high molecular weight carriers.Such high molecular weight carriers include, but are not limited to,polyalkylene glycol and polyethylene glycol (PEG), which arebiocompatible polymers with a wide range of solubility in both organicand aqueous media^([see J. Wang et al., [48]]).

Alternatively, microparticles such as microcapsules or cationic lipidscan serve as the pharmaceutically acceptable carriers of this aspect ofthe present invention. As used herein, microparticles include liposomes,virosomes, microspheres and microcapsules formed of synthetic and/ornatural polymers. Methods for making microcapsules and microspheres areknown to the skilled in the art and include solvent evaporation, solventcasting, spray drying and solvent extension. Examples of useful polymerswhich can be incorporated into various microparticles includepolysaccharides, polyanhydrides, polyorthoesters, polyhydroxides andproteins and peptides.

Liposomes can be generated by methods well known in the art^([52]).Alternatively, the nucleic acid molecules of this aspect of the presentinvention can be incorporated within microparticles, or bound to theoutside of the microparticles, either ionically or covalently

Cationic liposomes or microcapsules are microparticles that areparticularly useful for delivering negatively charged compounds such asthe nucleic acid molecules of this aspect of the present invention,which can bind ionically to the positively charged outer surface ofthese liposomes. Various cationic liposomes are known to be veryeffective at delivering nucleic acids or nucleic acid-protein complexesto cells both in vitro and in vivo, as reported^([53]).

Cationic liposomes or microcapsules can be generated using mixturesincluding one or more lipids containing a cationic side group in asufficient quantity such that the liposomes or microcapsules formed fromthe mixture possess a net positive charge which will ionically bindnegatively charged compounds. Examples of positively charged lipidswhich may be used to produce cationic liposomes include the aminolipiddioleoyl phosphatidyl ethanolamine (PE), which possesses a positivelycharged primary amino head group; phosphatidylcholine (PC), whichpossess positively charged head groups that are not primary amines; andN[1-(2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA).

As mentioned hereinabove the pharmaceutical compositions of this aspectof the present invention may further include excipients. The term“excipient” refers to an inert substance added to a pharmaceuticalcomposition to further facilitate administration of an activeingredient. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Various techniques for formulation and administration of drugs may befound in the opened literature^([54]). Suitable routes of administrationmay, for example, include oral, transmucosal, especially transnasal,parenteral delivery, including intramuscular, subcutaneous andintramedullary injections as well as intranasal, or intraocularinjections. Alternately, one may administer a preparation in a localrather than systemic manner, for example, via injection of thepreparation directly into the lungs.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes. Pharmaceuticalcompositions for use in accordance with the present invention may beformulated in conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries, whichfacilitate processing of the active ingredients into preparations which,can be used pharmaceutically. The “proper” formulation is dependent uponthe route of administration chosen.

For injection, the active ingredients of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological saltbuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions, and the like, for oralingestion. Pharmacological preparations for oral use can also be madeusing a solid excipient, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries ifdesired, to obtain tablets or dragee cores. Suitable excipients are, inparticular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspoly vinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatine as well as soft, sealed capsules made ofgelatine and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g., carbon dioxide,or dichlorodifluoromethane, trichlorofluoromethane, ordichloro-tetrafluoroethane^([55]). In the case of a pressurized aerosol,the dosage unit may be determined by providing a valve to deliver ametered amount. Capsules and cartridges of, e.g., gelatin for use in adispenser may be formulated containing a powder mix of the compound anda suitable powder base such as lactose or starch.

The preparations described herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents^([55]).

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-solubleform^([55]). Additionally, suspensions of the active ingredients may beprepared as appropriate oily or water based injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such assesame oil, or synthetic fatty acids esters such as ethyl oleate,triglycerides or liposomes. Aqueous injection suspensions may containsubstances, which increase the viscosity of the suspension, such assodium carboxymethyl cellulose, sorbitol or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents that increasethe solubility of the active ingredients to allow for the preparation ofhighly concentrated solutions^([55]).

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use^([55]).

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients effective to prevent, alleviate or amelioratesymptoms of disease or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. For any preparation used in themethods of the invention, the therapeutically effective amount or dosecan be estimated initially from in vitro assays. For example, a dose canbe formulated in small animal models and such information can be used tomore accurately determine useful doses in herds.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be verified by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals^([56]). The data obtained fromthese in vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in herds. The dosage may varydepending upon the dosage form employed and the route of administrationutilized.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribingveterinarian, etc.

Compositions including the preparation of the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

Pharmaceutical compositions of the present invention may, if desired, bepresented in a pack or dispenser device, such as an FDA approved kit,which may contain one or more unit dosage forms containing the activeingredient. The pack may, for example, comprise metal or plastic foil,such as a blister pack. The pack or dispenser device may be accompaniedby instructions for administration. The pack or dispenser may also beaccommodated by a notice associated with the container in a formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the compositions or human or veterinaryadministration. Such notice, for example, may be a labeling approved bythe U.S. Food and Drug Administration for veterinary prescription drugsor of an approved product insert.

To enable cellular expression of DNA nucleic acid molecules of thepresent invention, the nucleic acid construct of the present inventionfurther includes at least one “cis acting regulatory element”. As usedherein, the phrase “cis acting regulatory element” refers to apolynucleotide sequence, preferably a promoter, which binds atrans-acting regulator and regulates the transcription of a codingsequence located downstream thereto.

Any available promoter can be used by the present methodology. Preferredpromoters for use in aptamer expression vectors include the pol IIIpromoters such as the human small nuclear U6 gene promoter and tRNA genepromoters^([57]). It will be appreciated that many pol III promoters areinternal and are located within the transcription unit such that pol IIItranscripts include promoter sequences. To be useful for expression ofaptamer molecules, these promoter sequences should not interfere withthe structure or function of the aptamer. Therefore a preferred RNA polIII RNA promoter is the U6 gene promoter that is not internal^([58]).Suitable pot III promoter systems useful for expression of aptamermolecules are well described in the opened literature^([59]). It will beappreciated that promoters from the host cell or related species alsocan also be used.

The constructs of the present methodology preferably further include anappropriate selectable marker and/or an origin of replication.Preferably, the construct utilized is a shuttle vector, which canpropagate both in E. coli (wherein the construct comprises anappropriate selectable marker and origin of replication) and becompatible for propagation in cells, or integration in a tissue ofchoice. The construct according to the present invention can be, forexample, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus oran artificial chromosome.

Preferably, cationic lipids are used in combination with a neutral lipidin equimolar amounts as described hereinabove. Neutral lipids of use intransfection complexes include, for example, dioleoylphosphatidylethanolamine (DOPE) or cholesterol.

Typically a lipid mixture is prepared in chloroform, dried, andrehydrated in, e.g., 5% dextrose in water or a physiologic buffer toform liposomes. The resulting liposomes are mixed with a nucleic acidsolution with constant agitation to form the cationic lipid-nucleic acidtransfection complexes^([56-57]).

Prior to, concomitant with or following providing the nucleic acidmolecule of the present invention an agent can be provided to thesubject. An agent can be a molecule that facilitates prevention ortreatment of M. hyopneumoniae infection or clinical conditionsassociated with PRRS infection such as pneumonia. Examples of agents,according to this aspect of the present invention include, but are notlimited to, immunomodulatory agents (e.g., synthetic antibodies),antibiotics, antiviral agent (e.g., amantidine), antisense molecules,ribozymes and the like.

The antibody-like nature (i.e., specific binding to a polypeptidetarget) of the nucleic acid molecules of the present invention, allowsthe agents described hereinabove to be specifically targeted to aninfectious tissue upon attachment to the administered nucleic acidmolecule. For example an antisense molecule directed at a M.hyopneumoniae polypeptide (further described in the background section)can be targeted using the aptameric sequences of the present invention.“Chimeric” antisense molecules” are oligonucleotides that contain two ormore chemically distinct regions, each made up of at least onenucleotide. These oligonucleotides typically contain at least one regionwherein the oligonucleotide is modified so as to confer upon theoligonucleotide increased resistance to nuclease degradation, increasedcellular uptake, and/or increased binding affinity for the targetpolynucleotide. An additional region of the oligonucleotide may serve asa substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.

Chimeric antisense molecules of the present invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, as described above. Representative U.S. patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; 5,700,922, each of which is herein fully incorporated byreference.

Alternatively a ribozyme sequence can be targeted using the nucleic acidmolecules of the present invention. Ribozymes are being used for thesequence-specific inhibition of gene expression by the cleavage ofmRNAs. Several ribozyme sequences can be fused to the oligonucleotidesof the present invention.

Optionally, “DNAzymes” can be targeted using the methodology of thepresent invention. DNAzymes are single-stranded, and cleave both RNA. Ageneral model (the “10-23” model) for the DNAzyme has been proposed.“10-23” DNAzymes have a catalytic domain of 15 deoxyribonucleotides,flanked by two substrate-recognition domains of seven to ninedeoxyribonucleotides each. This type of DNAzyme can effectively cleaveits substrate RNA at purine:pyrimidine junctions^([60]).

Examples of construction and amplification of synthetic, engineeredDNAzymes recognizing single and double-stranded target cleavage siteshave been disclosed in U.S. Pat. No. 6,326,174.

Methods of nucleic acid-lipid coupling are well known in the art anddescribed in U.S. Pat. No. 5,756,291. For example, patent application WO2004076621 A2 describes a variety of methods for formation of conjugatesbetween nucleotide sequences and chelating agents; the chelating agentis joined to the nucleotides sequence by either a covalent bond or alinking unit derived from a polyvalent functional group. Thus, theaptamers or modified aptamers of the invention may be used alone intherapeutic applications or may be used for targeting agents to deliverpharmaceuticals or toxins to desired targets.

Detection of M. hyopneumoniae in Biological Samples

The ability of the nucleic acid molecules of the present invention tospecifically bind polypeptides of the M. hyopneumoniae allows the usethereof in diagnostic applications. To date, a number of tests areavailable for the diagnosis of M. hyopneumoniae infection. A traditionalapproach for identifying M. hyopneumoniae in biological samples involvescell culturing, thereby providing highly sensitive and specificdetection of viral infection. However, this approach is significantlylimited by the time required for cell culturing and identification of M.hyopneumoniae can range between 2 and 8 weeks, thus making itineffective in guiding the physician to an appropriate therapy. Since M.hyopneumoniae infection is normally self-limited, diagnosis must berapid if therapy is to be effective. Thus, cell culture methods are usedonly for providing retrospective epidemiological information.

Other Mycoplasma hyopneumoniae diagnostic methods include the use ofmonoclonal immunofluorescence assays and enzyme-linked immunoassay.However, not only are these methods limited to the identification oftype A M. hyopneumoniae infection, but they require considerabletechnical expertise, and result in high levels offalse-negatives^([22]). While accurate detection of MH by PCR has beenreported^([22-26]), it was found that the ability to detect various MHfield isolates differed significantly, and this was attributed togenetic differences among the isolates. This observation suggests thatfurther research is required to determine the accuracy of detection ofMH by PCR under field conditions.

Thus, according to yet another aspect of the present invention there isprovided a method of identifying M. hyopneumoniae in a biologicalsample.

As used herein a biological sample refers to any body sample such asblood, pleural fluid, respiratory fluids and nasal aspirates. Methods ofobtaining body fluids from vertebrates are well known in the art.

The method is implemented by contacting the biological sample with anucleic acid molecule including a polynucleotide sequence capable ofspecifically binding an M. hyopneumoniae polypeptide, describedhereinabove.

The nucleic acid molecules of the present invention can be attached to asolid substrate, such as described here in below.

Contacting is carried-out under conditions that allow the formation of apolypeptide-nucleic acid molecule duplex. Duplexes are preferably washedto remove any non-specifically bound polypeptides allowing only thosenucleic acid molecules specifically bound within the complexes to bedetected.

Polypeptide-bound nucleic acid molecules in the biological sample aredetected to thereby identify the Mycoplasma hyopneumoniae infection.

In general monitoring of polypeptide-nucleic acid molecule complexes iswell known in the art and may be carried-out as described hereinabove.These approaches are generally based on the detection of a label ormarker, such as described here in below. Preferably, detection of aninfected sample is effected by comparison to a normal sample, which isnot infected with the M. hyopneumoniae.

The Method of Generating the Nucleic Acid Sequence

To generate the nucleic acid molecules of the present invention, arobust selection approach is preferably employed.

Thus, according to an additional aspect of the present invention thereis provided a description of the method used to generate nucleic acidmolecules capable of inhibiting M. hyopneumoniae infection.

The method is implemented as follows. First, a plurality of nucleic acidmolecules are contacted with a polypeptide target, which participates inM. hyopneumoniae infection of cells as described hereinabove. Followingduplex formation (i.e., a non-Watson Crick complementation between thepolypeptide target and the nucleic acid molecules), at least one nucleicacid molecule of the plurality of nucleic acid molecules that is capableof specifically binding the polypeptide is identified. Finally,polypeptide bound nucleic acid molecules are isolated to therebygenerate the molecule that is capable of inhibiting M. hyopneumoniaeinfection.

Double stranded DNA molecules can be generated from a library ofoligonucleotide sequences including a randomized polynucleotide sequenceflanked by two defined nucleotide sequences that can be used forpolymerase chain reaction (PCR) primer binding. The library is amplifiedto yield double-stranded PCR products. The randomized sequences can becompletely randomized (i.e., the probability of finding a base at anyposition being 1:4) or partially randomized (i.e., the probability offinding a base at any position is selected at any level between 0-100%).

For preparation of single stranded aptamers, the downstream primer isbiotinylated at the 5′ end and PCR products are applied to an avidinagarose column. Single stranded DNA sequences are recovered by elutionwith a weakly basic buffer. Single stranded RNA molecules can begenerated from an oligonucleotide sequence library, which is amplifiedto yield double-stranded PCR products containing a T7 bacteriophagepolymerase promoter site. RNA molecules can then be produced by in vitrotranscription using T7 RNA polymerase.

The nucleic acid molecules of this aspect of the present invention canbe generated from naturally-occurring nucleic acids or fragmentsthereof, chemically synthesized nucleic acids, enzymatically synthesizednucleic acids or nucleic acid molecules made by a combination of theforegoing techniques

The library of this aspect of the present invention is generatedsufficiently large to provide structural and chemical coverage ofselected nucleic acid modifications described hereinabove.

Typically, a randomized nucleic acid sequence library according to thisaspect of the present invention includes at least 10¹⁴ sequencevariants.

Nucleic acid modifications can be effected prior to incubation with thetarget polypeptide. In this case, although screening is implemented onthe final modified aptamer, modification is restricted not to interferewith any process, such as an enzymatic process (e.g., transcription),which takes place during the screening.

Alternatively, a nucleic acid molecule can be modified followingselection (i.e., isolation of a polypeptide bound nucleic acidmolecule). Thus, a wide range of functional groups can be usedsimultaneously. In this case, electrospray ionization mass spectrometry(ESI-MS) can be used to elucidate the right functional group.

In any case, once nucleic acid molecules are obtained they are contactedwith the polypeptide target, as mentioned hereinabove.

Incubation of the nucleic acid molecules with the target polypeptide ofthis aspect of the present invention is preferably implemented underphysiological conditions. As used herein the phrase “physiologicalconditions” refers to salt concentration and ionic strength in anaqueous solution, which characterize fluids found in the metabolism ofvertebrate animal subjects which can be infected with M. hyopneumoniae,also referred to as physiological buffer or physiological saline. Forexample physiological fluids of swine are represented by anintracellular pH of 7.1 and salt concentrations (in mM) of sodium 3-15;potassium 140; magnesium 6.3 Calcium 10-4; Chloride 3-15, and anextracellular pH of 7.4 and salt concentrations (in mM) of sodium 145;potassium 3; Magnesium 1-2; Calcium 1-2; and Chloride 110.

The nucleic acid molecules can be incubated with the target polypeptideeither in solution or when bound to a solid substrate.

It will be appreciated that some of the above-described basemodifications can be used as intermediates for attaching the nucleicacid molecule to a solid substrate. For example, the modified uridineshown in group 48 of FIG. 5 c , can serve as a common intermediate whichmay be further modified by substitution of the imidazole with a widevariety of hydrophobic, hydrophilic, charged and cross linking groups,prior to activation as the phosphoramidite reagent used in solid phasesynthesis

Methods for attaching nucleic acid molecules to solid substrates areknown in the art including but not limited to glass printing,photolithographic techniques, inkjet printing, masking and the like.

Typically, a control sample is included to select against nucleic acidmolecules that bind to non-target substances such as the solid supportand/or non-target peptides/proteins.

Separation of unbound nucleic acid sequences and identification of boundnucleic acid sequences can be effected using methods well known in theart. Examples include, but are not limited to, selective elution,filtration, electrophoresis and the like.

Alternatively, imaging can identify bound aptameric molecules. Forexample, optical microscopy using bright field, epifluorescence orconfocal methods, or scanning probe microscopy can be used to identify apolypeptide bound nucleic acid molecule. To facilitate visualization,nucleic acid molecules or polypeptides are preferably labeled using anyradioactive, fluorescent, biological or enzymatic tags or labels ofstandard use in the art^([61]).

The following illustrates a number of labeling methods suitable for usein the present invention. For example, nucleic acid molecules of thepresent invention can be labeled subsequent to synthesis, byincorporating biotinylated dNTPs or rNTP, or some similar means (e.g.,photo-cross-linking a psoralen derivative of biotin to RNAs), followedby addition of labeled streptavidin (e.g., phycoerythrin-conjugatedstreptavidin) or the equivalent. Alternatively, fluorescent moieties areused, including but not limited to fluorescein, lissarine,phycoerythrin, rhodamine, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, FluorX andothers.

It will be appreciated that the intensity of signal produced in any ofthe detection methods described hereinabove may be analyzed manually orusing a software application and hardware suited for such purposes.

Isolation of an aptamer sequence (i.e., polypeptide-bound nucleic acid)typically involves sequence amplification such as by PCR. Amplificationmay be conducted prior to, concomitant with or following separation fromthe target polypeptide. The PCR method is well known in the art^([62]).It will be appreciated that if RNA molecules are used, the amplified DNAsequences are transcribed into RNA.

The recovered nucleic acid molecule, in the original single-stranded orduplex form, can then be used for iterative rounds of selection andamplification (i.e., target polypeptide binding). Typically, followingthree to six rounds of selection/amplification, nucleic acid moleculesthat bind with a preferred affinity of nM to M range can be obtained.

It will be appreciated that methods for identifying nucleic acidmolecules capable of specifically binding polypeptide targets are knownin the art^([27]). For example, U.S. Pat. No. 5,270,163, incorporated byreference in its entirety, discloses the SELEX method for theidentification of nucleic acid ligands as follows. A candidate mixtureof single-stranded nucleic acids having regions of randomized sequenceis contacted with a target compound and those nucleic acids having anincreased affinity to the target are partitioned from the remainder ofthe candidate mixture. The partitioned nucleic acids are amplified toyield a ligand-enriched mixture. The target-oligonucleotide complexesare then separated from the support and the uncomplexedoligonucleotides, and the complexed oligonucleotides are recovered andsubsequently amplified using PCR. The recovered oligonucleotides may besequenced and subjected to successive rounds of selection usingcomplexation, separation, amplification and recovery.

Alternatively, the nucleic acid sequences of the present invention canbe generated by rational drug design. Rational drug design is theinventive process of finding new medications based on the knowledge of abiological target[^(63]). The drug is most commonly an organic smallmolecule that activates or inhibits the function of a biomolecule suchas a protein, which in turn results in a therapeutic benefit. Thus,rational drug design is a potent means of identifying small moleculesthat are complementary in shape and charge to the biomolecular targetwith which they interact and therefore will bind to it.

Alternatively, a refined aptamer sequence can be elucidated by modifyinga known aptamer structure (e.g., APT3Cooc10)) using a softwarecomprising “builder” type algorithms which utilizes a set of atomiccoordinates defining a three dimensional structure of the binding pocketand the three-dimensional structures of the basic aptamer tocomputationally assemble a refined aptamer. Ample guidance forperforming rational drug design via software employing such “scanner”and “builder” type algorithms is available in the literature of theart^([64]).

Criteria employed by software programs used in rational drug design toqualify the binding of screened aptamer structures with binding pocketsinclude gap space, hydrogen bonding, electrostatic interactions, van derWaals forces, hydrophilicity/hydrophobicity, etc^([65]). Generally, thegreater the contact area between the screened molecule and thepolypeptide binding region, the lower the steric hindrance, the lowerthe “gap space”, the greater the number of hydrogen bonds, and thegreater the sum total of the van der Waals forces between the screenedmolecule and the polypeptide binding region of, the greater will be thecapacity of the screened molecule to bind with the target polypeptide.The “gap space” refers to unoccupied space between the van der Waalssurface of a screened molecule positioned within a binding pocket andthe surface of the binding pocket defined by amino acid residues in thebinding pocket. Gap space may be identified, for example, using analgorithm based on a series of cubic grids surrounding the dockedmolecule, with a user-defined grid spacing, and represents volume thatcould advantageously be occupied by a modifying the docked aptamerpositioned within the binding region of the polypeptide target. Contactarea between compounds may be directly calculated from the coordinatesof the compounds in docked conformation using various commerciallyavailable Molecular Structure (MS) software packages.

In any case, once putative aptamer sequences are identified they areexamined for specific binding to the target polypeptide, which can beimplemented using a number of biochemical methods known in theart^([66]).

Alternatively or additionally, the nucleic acid sequences of the presentinvention are tested for inhibiting M. hyopneumoniae infection in vitrosuch as with pulmonary ciliated epithelial cell line, or in vivo asfurther described in Example 2 (in vitro) and Example 3 (in vivo) of theExamples section.

As mentioned hereinabove, the nucleic acid sequences of the presentinvention can be used for treating M. hyopneumoniae infection.

Thus according to yet a further aspect of the present invention there isprovided a method of treating M. hyopneumoniae infection.

The method is implemented by providing to a subject in need thereof, atherapeutically effective amount of the polynucleotide and/or syntheticantibody of the present invention, described hereinabove.

Preferred administration routes and pharmaceutical compositions aredescribed hereinabove.

It will be appreciated that synthetic antibodies generated according tothe teachings of the present invention can be used also for identifyingM. hyopneumoniae in a biological sample.

The method can be implemented by contacting a biological sample such asdescribed hereinabove, with the synthetic antibody of the presentinvention. Thereafter, immunocomplexes including the synthetic antibodyin the biological sample are detected, to thereby identify the M.hyopneumoniae strain in the biological sample.

The nucleic acid molecules, conjugates thereof, polynucleotides, andsynthetic antibodies generated according to the teachings of the presentinvention can be included in a bio-detection device or a diagnostic kitor therapeutic kit. These reagents can be packaged in one or morecontainers with appropriate buffers and preservatives and used fordiagnosis or for directing therapeutic treatment.

Thus, nucleic acid molecules and conjugates thereof can be each mixed ina single container or placed in individual containers. Preferably, thecontainers include a label. Suitable containers include, for example,bottles, vials, syringes, and test tubes. The containers may be formedfrom a variety of materials such as glass or plastic.

In addition, other additives such as stabilizers, buffers, blockers andthe like may also be added. The nucleic acid molecules and conjugatesthereof of such kits can also be attached to a solid support, asdescribed and used for diagnostic purposes.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature^([67]). The procedures thereinare believed to be well known in the art and are provided for theconvenience of the reader. All the information contained therein isincorporated herein by reference.

Example 1 the Peptidic P97 Complex Specific Aptamers Rationale andDesign

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) wasimplemented in order to identify aptamer oligonucleotides that bind theM. hyopneumoniae.

Materials and Experimental Procedures

Library Generation—The aptamer library containing a central randomizedsequence of 40 nucleotides flanked by a common 5′ sequence—AAT TAA CCCTCA CTA AAG GG, denoted as T3) (SEQ ID NO. 21) and a common 3′sequence—5′-TAT GGT CGA ATA AGT TAA-3′ (SEQ ID NO. 22) was synthesizedin a 380B DNA synthesizer. The library included a 30 nucleotides randomsegment, over all 10¹⁶ molecules and generated according tomanufacturer's instructions.

SELEX—ssDNA aptamers were denatured at 80° C. for 10 min and then cooledon ice for 10 min. Aptamers 30 nmole were mixed with 25 μg of P97peptidic complex (further described herein below) in 500 μl selectionbuffer (50 mM Tris-HCL; pH 7.4, 5 mM KCl, 100 mM NaCl, 1 mM MgCl₂ tRNA,0.2% BSA) at 37° C. for 30 min. Aptamer-peptide complex was purified byadding the 25 μl Ni-NTA superflow (Qiagen) and amplified by PCR usingprimers directed to the common sequences in the aptamer library, i.e.,5′-AAT TAA CCC TCA CTA AAG GG-3′, (T3) (SEQ ID NO: 21) and 3′ primer 5′TTA ACT TAT TCG ACC ATA-3′ (SEQ ID NO: 23). SELEX was repeated 3 times,following which amplified nucleotides were transformed into E. coli. PCRconditions for SELEX included 5 min 95° C./1 min 95° C./1 min 55° C./1min 72° C./10 min 72° C. and 100 pmole of each primer.

Reverse Screening of Aptamer—Selected ssDNA molecules from eachindividual clone were biotinylated using the B-T3 which is same sequencewith 5′ primer (T3 primer), and Klenow fragment (2 unit/ml). To preparesingle stranded biotin conjugated APT3Cooc10 aptamer for the reversescreening. T3 Primer was Biotin labelled. A 96-well flat bottom ELISAplate was prepared by coating each well with 100 μl of streptavidin (100μg/ml) diluted in 0.1 M NaHCO₃ following by a 37° C. overnightincubation. Following several washings with PBS, wells were blocked with200 μl of PBS containing 1% BSA for 2 hours at room temperature andsubsequent washing three times with PBS-T (10 mM PBS containing 0.05%(v/v) Tween-20). Thereafter, 100 μl of 2.5 pmole/100 μlbiotinylated-ssDNA were added to the wells and incubated at 37° C. for 2hours followed by washing four times with PBS-T. T3 primer was used asnegative control. Following washing, 100 μl of fluid containing 2 μghistidine labelled P97 peptidic complex were added to the indicatedwells and incubated at 37° C. for 2 hours. The wells were then washedfor 4 times with PBS-T. The reverse screening assay was completed usingthe DAKO Mycoplasma hyopneumoniae ELISA tests (K004311-9 kit, Oxoid Ltd,UK).

Enzyme-Linked Immunosorbent Assay (ELISA)—The ELISA tests werecarried-out following all the guidelines provided with the DAKO kit.Reaction was terminated with 100 μl of 0.5M H₂SO₄, and the plates wereread with a multichannel spectrophotometer at 450 nm.

Results—In order to identify oligonucleotides that bind to the peptidiccomplex P97, a nucleotide library containing random 30 nucleotidesbetween conserved linkers, was synthesized. The library included 10¹⁶types of different ssDNA, which were hybridized to the P97 peptidiccomplex and purified by Ni-NTA resin. Following purification, ssDNAswere amplified by PCR using the linker sequences. The process wasrepeated 4 times and re-screening of the P97 complex was implemented byELISA. This reverse-screening process resulted in three oligonucleotideaptamers denoted as “APT3Cooc10”, “APT3Histo1”, and “APT3Cooc3”. Allthree aptamers showed similar binding capacity to the P97 complex (FIG.6 ). Note a significant protection of APT3-Cooc10 and APT3Cooc3 againstthe intact virus as compared to control nucleic acid is notable (p=0.042and p=0.0008, respectively), and a significant reduction in APT3-Cooc10binding to the intact virus as compared to the APT3-Cooc3 aptamer(p=0.017). Therefore, structural and functional analysis of theAPT3Cooc10 and APT3Cooc3 oligonucleotides only was further implemented.Proposed secondary structures using DNA draw program for APT3Cooc10,APT3Histo1, and APT3Cooc3, as well as a control oligonucleotide areshown in FIGS. 4 a, e, c, respectively.

Example 2. In-Vitro Aptamer Protection from M. hyopneumoniae Infection

The protective effect of the APT3Cooc10 aptamer against two virulentstrains of M. hyopneumoniae was investigated in vitro using cilia ofpulmonary epithelium cells (PEC).

Materials and Experimental Procedures

Mycoplasma hyopneumoniae—M. hyopneumoniae 232 (GenBank accessionAE017332) and M. hyopneumoniae 7448 (GenBank accession AE017244) wereobtained from the faculty of Veterinary Microbiology and PreventiveMedicine, Iowa State University, Ames, Iowa 50111, USA (Prof. F. C.Minion). The cells were grown in modified Friis medium at 37° C. andharvested during the exponential phase when the medium became honeycoloured and pH was in the range 6.9-7.2. Mycoplasma cells wereharvested by centrifugation at 10 000 g for 20 min and the pellets werewashed twice with Tris-buffered saline (TBS: 10 mM Tris/HCl, 150 mMNaCl; pH 7.5) and the final pellets were frozen a −20° C. until use.When used, the pellet of Mycoplasmas was re-suspended to 1/10 of theoriginal volume in an adherence buffer (AB) that consisted of a RPMI1640 medium containing 1% gelatin.

Cells, and cilia extraction—Pulmonary Epithelium Cells (PEC) wereobtained from RTI LLC (Brookings, S. Dak.) and maintained in Dulbecco'smodified Eagle's medium (DMEM) supplemented with heat inactivated 10%fetal calf serum (FCS). The cilia were extracted and purified from theharvested PEC using the method described by Zhang et al.^([20]). Inbrief, ciliated cells were suspended in 40 ml of buffer containing 20 mMTris, 10 mM EDTA, and 125 mM sucrose (TES buffer [pH 7.2]). Two washingswith TES buffer were conducted by centrifugation at 300×g for 5 min. Thecell pellet was re-suspended in 6 ml of AES buffer (80 mM acetate, 10 mMEDTA, 125 mM sucrose [pH 6.8]) and incubated for 5 min at 25° C. Then,0.2 M CaCl was added to a final concentration of 10 mM. The mixture wasvortexed for 10 min, diluted with 20 ml of TES buffer, and thencentrifuged at 500×g for 10 min. The sediment, which contained mainlycell bodies, was discarded, and the supernatant, containing cilia, washarvested by centrifugation at 18,000×g for 15 min at 40° C. The ciliarypellet was washed twice with phosphate buffered saline (PBS) and storedat −80° C. The purity of the cilia was ascertained by light microscopy.Protein concentrations were determined with the bicinchoninic acidprotein assay reagent (Pierce™ BCA Protein Assay Kit 23225, ThermoScientific, Rockford, Ill.) according to the instructions provided withthe product. Porcine albumin (fatty acid free; 10 μg/ml) and gelatin (10μg/ml) were used to coat plates as negative controls under theconditions used for cilia.

Coating of plates, and cilia binding Assay—Purified cilia (2 mg/ml inPBS) were solubilized with sodium dodecyl sulfate (SDS; 1 mg/mg ofprotein) at 37° C. for 45 min. This preparation was further diluted withsodium carbonate buffer (0.1 M; pH 9.5) to a final concentration of 10μg of protein per ml. To each well of flat-bottom microtiter plates(Sarstedt, model 83.3824) was added 100 μl of this solution (1 μg ofcilia per well) and incubated for 3 h at 37° C. Note that thecilia-coated plates can be stored at −80° C. for a long period (>6months) without the loss of adherence^([20]).

After four washings with PBS (pH 7.4), the cilia-coated plates wereblocked with 200 μl of AB per well for 2 h at 37° C. Then, 100 μl ofMycoplasma hyopneumoniae cells re-suspended in AB was added to eachwell, and the plates were incubated at 37° C. for 90 min. Non-adherentMycoplasmas were removed by four washings with PBS. Subsequently, 100 μlof rabbit antibodies to M. hyopneumoniae (Biorbyt, Calif.) was added andincubated at 37° C. for 60 min. The wells were washed four times withPBS and then incubated with 100 μl of goat anti-rabbit peroxidaseconjugate (Abcam, Mass.) for 60 min at 37° C. After four washings withPBS, 100 μl of peroxidase substrate[2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS)] (from KPLInc. Gaithersburg, Md.) was added to each well and incubated at 25° C.for 10 min. Optical densities (OD) at 405 nm were measured with anautomated microplate reader (model ELx808IU; Bio-Tek Instruments, Inc.,VT).

For the aptaneutralization assays, various aptamers diluted toappropriate concentrations in AB were mixed with Mycoplasmas and addedto the cilium-coated plates. After incubation and washings, the bindingof Mycoplasmas to the plates was detected with rabbit anti-M.hyopneumoniae antibodies and goat anti-rabbit conjugates as describedabove. To optimize the sensitivity to the neutralization, as suggestedby Zhang et al. 2.5×10⁸ CCU of Mycoplasmas was used in each well so thatthe OD of the controls was about 0.5˜0.6.

Two approaches were used to study neutralization/inhibition.

-   (i) The aptamers were pre-incubated with Mycoplasmas at 37° C. for 1    hr, and the incubated Mycoplasmas+aptamers were applied to the    cilium-coated plates.-   (ii) Cilium-coated plates were pre-incubated with the aptamers at    37° C. for 1 h, unbound aptamers were removed by four washings, and    the Mycoplasmas were added to the plates.

Thereafter, procedures for both approaches were identical, including thesequential addition of antibodies to Mycoplasmas, secondary antibodyconjugates, and peroxidase substrates. The percent neutralization wascalculated as follows:Percent neutralization=(OD from AB−OD from an aptamer)/OD value from AB.

The IC₅₀, representing the concentration of an aptamer that resulted in50% inhibition, was determined from the neutralization kinetics obtainedwith multiple concentrations of the aptamer.

Results—The aptamer ability to prevent the adhesion of M. hyopneumoniaeonto the cilia of the pulmonary cells was tested. Typical results areshown in FIG. 7 , where cells treated with aptamer APT3Cooc10 prior toM. hyopneumoniae infection demonstrated a significant reduction inMycoplasma binding to the cilia.

Mycoplasma hyopneumoniae bound specifically to cilium-coated wells butnot to control wells coated with gelatin or porcine albumin (FIG. 7 ).Note that Mycoplasma only binds to the cilia-coated plates and that thegelatin-coated plates are a very effective control for the bindingassays. ODs obtained with the control wells (gelatin-coated) were alwaysless than 0.10. As expected, the number of Mycoplasmas influenced thedegree of binding. The concentration of cilia for coating the plates was1 ug of protein per well. About 4×10⁸ CCU of Mycoplasmas was requiredfor half-maximal binding at 37° C. while about 10 CCU of Mycoplasmas perwell was required to reach saturated binding.

Ten aptamers that bind to the P97 peptide have been identified. Three ofthem, APT3Cooc3, APT3Cooc10, and APT3Histo1, bound to the ligands on thesurface of Mycoplasmas and therefore are receptor analogs. Since thebinding of Mycoplasma onto the ciliated-plate is dependent on theconcentration of Mycoplasma that is added to the wells (see FIG. 7 ),the quantity of aptamers that is needed to neutralize a givenconcentration of Mycoplasma is also concentration-dependent FIG. 8 showsthat about 2000 pmole of APT3Coo10 are required to prevent the adhesionof 4×10⁸ CCU/well of Mycoplasma onto 1 μg/well of cilia. FIG. 8 alsodemonstrates that the aptamers do not bind, or modify the cilia and theciliated cells.

The ability of APT3Cooc10 to prevent the adhesion of Mycoplasma onto thepulmonary ciliated cells is evident (see FIG. 8 ) and shows thatAPT3Cooc10, APT3Cooc3, and APT3Histo1 can be used to prevent or stop thepropagation of Mycoplasma into the luminal surface of tracheas, bronchi,and bronchioles of swine. Furthermore, swine being exposed to nebulizedparticles containing any of these three aptamers will not have anydetrimental or negative effects. As shown in FIG. 8 , the highestprotective effect was achieved using APT3-Cooc10 at the concentrationrange of 1000 to 4000 pmole.

Example 3 In-Vivo Aptamer Protection from M. hyopneumoniae Infection

The protective effect of the APT3Cooc10 aptamer against M. hyopneumoniaeinfection (the 232 strain) was determined using an experimental protocolsimilar to the one used by D. A. Murphy et al^([68]) for comparisonpurposes. In this older study, it was found that aerosolized commercialvaccine did not offer any protection against Mycoplasma hyopneumoniaeinfection and only limited protection with the conventionalintramuscular injection. The main difference between the two studiesbeing that the commercial aerosolized vaccine was replaced by nebulizedAPT3Cooc10 aptamers.

Materials and Experimental Methods.

Swine—Twenty-one American Yorkshire swine 8 weeks-old castrated malepigs were obtained from a closed commercial farm (M.-hyopneumoniae-freeas determined by repeated slaughter examination and serologic testing)and were randomly selected from 7 litters. The pigs were fed a normalgrower ration that did not contain antibiotics.

Experimental design—The pigs were randomly assigned by litter and bodyweight to 1 of 3 treatment groups. The first group (PBS group) served asthe negative-control, and was exposed to nebulized PBS. The second group(APT3 group) was exposed to nebulized aptamer solutions. The third group(commercial vaccine/CV group) was vaccinated using a commerciallyavailable injectable vaccine according to the manufacturer's labeldirections. Two weeks after the last aerosol vaccination, all pigs werechallenge-exposed by intra-tracheal inoculation. Four (4) weeks afterthe challenge exposure, all pigs were necropsied and examined. Inparticular, pre-vaccination and post-vaccination serum were assayed forM. hyopneumoniae specific antibodies.

Vaccine—A commercially available injectable M. hyopneumoniae vaccine wasobtained (Suvaxyn®MH One, Zoetis Canada Inc. Available at the zoetis.cawebsite). The vaccine contains a proprietary water-soluble adjuvant.Pigs in the CV-group each received the recommended dose of 2 mladministered intramuscularly in the neck. A total of 2 doses were givenat 2-weeks intervals. Pigs in the APT3-group each received 4000 pmole ofAPT3Cooc10, which was mixed with 10 ml of PBS and administered bynebulization. A total of 3 doses were given at two-week intervals.

Nebulizing procedure—The aptamers and the PBS for the first two groupswere nebulized by use of a commercial nebulizer (PARI LC® Starnebulizer) that is producing 0.5-0 to 5 μm diameter aerosol particles(3.1 μm median size). Pure medical air was used with the nebulizer.Sterile re-usable chambers were used for nebulizing the APT3 and thePBS. The aerosol passed through a one-way valve from the nebulizationchamber into a flexible 1 meter long tube, and was delivered to the pigsthrough an anaesthetic veterinary mask (SurgiVet, model 32393B1). TheAPT3 aerosol was not irritating to the pigs and tranquilization was notrequired. Pigs were restrained in a padded sling during the aerosolprocedure. It tool 15 to 30 minutes for a pig to inhale its full dose ofAPT3 or PBS.

Challenge-exposure procedure—The pigs were challenged with M.hyopneumoniae 232 (GenBank accession AE017332) that was obtained fromthe faculty of Veterinary Microbiology and Preventive Medicine, IowaState University, Ames, Iowa 50111, USA (Prof. F. C. Minion). The cellswere grown in modified Friis medium at 37° C. and harvested during theexponential phase when the medium became honey coloured and pH was inthe range 6.9-7.2. Mycoplasma cells were harvested by centrifugation at10,000 g for 20 min and the pellets were washed twice with Tris-bufferedsaline (TBS: 10 mM Tris/HCl, 150 mM NaCl; pH 7.3) and the final pelletswere frozen a −20° C. until use. Each challenge group was inoculatedintra-tracheally with an M.-hyopneumoniae broth culture containing about10⁸ CCU/ml of MH232.

Clinical evaluation—Pigs were monitored twice daily while the stallswere being cleaned, for a minimum of 15 min for clinical signs ofillness, and observations were recorded on a scale of 0 (normal), 1(mild abnormal) or 2 (severe abnormal) for general health, mobility,appetite and coughing at each inspection. The cumulative (total)coughing scores were calculated for each pig and a mean score perinspection was also determined. Pigs were weighed 3 days prior tochallenge and again at 21 and 28 days post-challenge. Any pig thatcoughed more than once during that time was recorded as persistentlycoughing for that day. Pigs that would produce a short cough afterputting their snout into the feed were not counted.

Necropsy—The pigs were euthanatized by electrocution and exsanguination,and the lungs were removed quickly for evaluation. The lungs wereevaluated for macroscopic lesions and scored using the Goodwin lungscoring system^([8-9, 22, 39]). In addition, the percentage of affectedlung was calculated by weighing all grossly consolidated lesions andcomparing with the weight of the entire lung. The entire right lung waslavaged with four 50 ml aliquots of PBS. The total cells within thelavage fluid were counted manually by use of a hemocytometer. Theremaining fluid was centrifuged, and the supernatant was stored fordetermination of antibody titer.

Quantitative culture of mycoplasmas—The presence of M. hyopneumoniae inthe lung mycoplasmal fluid was determined by use of immunohistochemistry(IHC) assay. Immunohistochemical detection of M. hyopneumoniae-specificantigen on selected lung tissues was done using the heat-induced epitoperetrieval technique. Paraffin-embedded tissue sections were dewaxed andrehydrated, covered with 1:10 ethylenediaminetetraacetic acid buttersolution, pH 6.0, placed in a microwave, and boiled for 5 minutes. Aftercooling for 20 minutes, the slides were rinsed, and M. hyopneumoniaemonoclonal antibody (identification number D79DI-7 was applied in a1:500 dilution for 2 hours at room temperature and further processed byusing a labeled streptavidin-biotin detection kit (DAKO). Specificity ofthe M. hyopneumoniae IHC procedure was evaluated by testing sectionsfrom formalin-fixed paraffin-embedded blocks from pigs known positivefor other pathogens (SIV, PRRSV). Specificity was determined to be 100%.There was no evidence of cross-reaction with any of the pathogenstested. Sensitivity was evaluated by comparing the IHC results withthose obtained with M. hyopneumoniae culture, which is considered thegold standard for M. hyopneumoniae detection. Known M.hyopneumoniae-positive tissue sections as well as known M.hyopneumoniae-negative tissue sections were used as controls for eachIHC run. Slides were scored ranging from 0 to 3 (0, no signaldetectable; 1, weak labeling lining the ciliated epithelium of at leastone airway; 2, weak-to-moderate labeling on the surface of a low numberof airways; 3, intense labeling on the surface of several airways).

ELISA procedure—All serum samples were assayed for antibodies to M.hyopneumoniae using the IDEXX ELISA test kits and according to themanufacturer's instructions (IDEXX, M. hyo, Ab Test Part number:99-06733). Known positive and negative sera were included as controls ineach plate. Briefly, 100 ml of 1:40 diluted individual sample was addedinto the M. hyo antigen-coated ELISA plates. After incubation of thesample in the coated well and washing out any unbound material from thewells, goat anti-swine IgG conjugate was added to detect the boundantibodies. Test results were determined on the basis of thesample-positive (SIP) ratio: positive 5 S/P. 0.4, negative 5 S/P, 0.3,and suspect 5 S/P from 0.3 to 0.4. Although the sensitivity of theIDEXX-ELISA was considered high, the specificity of the assay was low.

Statistical analysis—The results for the 3-treatment groups werecompared using the Scheffé's method for multiple comparisons, withα=0.05. Logarithmic transformation was used to normalize the percentageof the pneumonia and ELISA data.

Results—Clinical observations: The aptamers delivered in aerosol (APT3group) did not cause any irritation to the pigs and did not causecoughing, sneezing or other signs of proximal or distal respiratorytrack irritation. Appetite did not appear to be affected, and rate ofgain was similar for pigs of all 3-treatment groups during this study.

Persistent coughing began in pigs of all 3-treatment groups about 2weeks after the challenge exposure. All pigs in the PBS and CV groupswere observed coughing over the next 2 weeks. Mean number of days ofpersistent coughing (4.6 and 3.7 days, respectively) was similar forboth groups. Only 4 of 7 pigs in the APT3 group were observed coughingduring the study. Additionally, mean numbers of days with persistentcoughing (1.0 day) was lower in the APT3 than in the CV group (see Table2).

Gross lesions of pneumonia between the PBS and the CV groups were 15.3%(means) and 5.3% (means) respectively. In contrast, gross lesions ofpneumonia were less severe in pigs of the APT3 group (mean 1.6% of lungaffected) than in pigs of the CV group (see Table 2).

TABLE 2 Clinical results of post-challenge exposure Weight PersistentLesion on gain cough lungs Titer Group # pigs (Kg) (day) (%) (CCU/g) PBS7 27.6 ± 3.2 4.6 ± 1.9 15.3 ± 10.3 10^(6.2) ± 1.4 CV 7 28.2 ± 3.1 2.7 ±1.2 5.3 ± 4.4 10^(4.3) ± 2.0 APT3 7 30.0 ± 2.8  1.2 ± 1.2^(a)  1.6 ±2.6^(a) 10^(3.7) ± 1.3 ^(a)Significantly different from the PBS-controlgroup with α = 0.05 (Scheffé's method)

Results—Mycoplasma titers at necropsy: All pigs were positive for M.hyopneumoniae by the IHC tests. Titer of pulmonary mycoplasmal organismswere similar in the PBS, (mean 10^(6.2) CCU; range 10^(4.8) to10^(7.6)), and in the CV-group (mean 10^(4.3) CCU; range 10^(2.3) to10^(6.3)). As shown in Table 2, they were systematically lower for theAPT3 group (mean 10^(3.7) CCU; range 10^(2.4) to 10^(5.0)).

Results—Antibodies (ELISA) responses to treatments: The antibody levelsof the mean pre-vaccination, post-vaccination, and post-challengeexposure to M. hyopneumoniae was determined for each treatment group(Table 3). The sera were assayed at a dilution of 1:5. This lowdilution, several pigs in each group had detectable antibody activityagainst M. hyopneumoniae prior to vaccination. Serum antibody was notincreased in either of the vaccinated groups 2 weeks after the lastaerosol vaccination (4 weeks after the last CV vaccination). Similarresults were obtained fin the study of Murphy et al.^([68]) (data notshown).

At necropsy (4 weeks after the challenge exposure), antibody wasdetected in serum of all pigs. Serum antibody activity in pigs of theCV-group (mean O.D. 1.1.89) was significantly higher than that in pigsof the PBS (0.759) and APT3 (0.663) groups. Indicating that the CVvaccination may have primed the pigs ability to immunologically respondto the MH232 challenge. However, the fact that antibody activity did notincrease after vaccination, and that post-vaccination antibody activityin the vaccinated CV-group pigs was not different from the control pigs,indicates that the Suvaxyn®MH One vaccine (CV-group) was inducing a weakantibody response.

Overall evaluation of aerosol vaccination—Under the conditions of thisstudy, the aerosol vaccination (APT3) did not induce a full protectiveimmunity in pigs. However, aerosol vaccination with the APT3Cooc10aptamers, perform much better than the “standard” intra-muscularvaccination with Suvaxvn®MH One (CV-group) that has been used for morethan 30 years. Weight gains were higher with the APT3 vaccination whilethe Mycoplasma titers, and the lung lesions were much lower with theAPT3 vaccination than with the CV. In fact, the markedly reducedpulmonary lesions with the APT3 vaccination confirmed the efficacy ofthe APT3Cooc10 used in this study. The primary function of the APT3aptamers is to bind the cilia of Mycoplasma hyopneumoniae, not totrigger the immune system. In fact, APT3Cooc10, APT3Cooc3, andAPT3Histo1 are aptamers and do not act as antigen, and have no effect onthe pulmonary epithelial cells (see FIG. 8 ).

As pointed-out by Murphy et al.^([68]), possible reasons to explain theresults for the aerosol APT3Cooc10 vaccine may include (i) failure ofthe aptamer to deposit in the lungs, (ii) rapid clearance of the aptamerfrom the lungs by the mucociliary apparatus, (iii) inadequatevaccination frequency, and (iv) inadequate dose of aptamers.

Location of aerosol particle deposition in the respiratory tract may bedependent on the size of the aerosol particles. Deposition of aerosol inbronchioles and alveoli is maximal when the aerosol droplets size is 0.5to 3 μm diameter. The medical nebulizer used in this study wasmanufactured to produce aerosol droplets in the range of 3.1 μm, whichis the upper side of the optimal range of particles to penetrate deepinside the pulmonary alveoli.

Although clearance of mycoplasmal vaccine from the respiratory tract ofpigs was not measured in this study, clearance of microbial organismfrom the respiratory tract is known to be a remarkably rapidprocess^([69]). The mucociliary mechanism of clearance might especiallydiminish the immune response to the larger particulates antigen in theupper respiratory tract. The local immune response to intra-nasallyadministered particulate antigen (e.g. sheep RBC) in rats is well-knownto be lower than the local immune response to intra-nasally administeredsoluble antigen (e.g. lipopolysaccharide), suggesting that particulateantigen might be more easily entrapped in the mucus layer beforecontacting the underlying epithelial cells^([70]). Rapid clearance ofantigen from the respiratory tract might reduce the total antigenic massexposed to the immune system, and might reduce the duration of antigenicexposure. In other words, the APT3Cooc10 aptamers were cleared from therespiratory tract before having the opportunity to meet and bind withthe MH232. On the other hand, the same clearance mechanism might havehelped to remove the MH232 cells that have been neutralized by theAPT3Cooc10 aptamers, and this would explain the lower titers of MH232observed in the APT3 group (compared to CV and PBS groups).

The optimal time interval for aerosol vaccine administration may beobtained using methodologies known to those skilled-in-the-art. Inprevious studies of a variety of vaccines in a variety of species,aerosol vaccines have been administered once^([69]), or were repeated at7, 14, and to 28 days intervals^([68-71]). In general, repeatedvaccination at a 14-day interval resulted in an increase clearance ofthe virulent pathogen. But repeating the vaccination at an interval of20 to 40 days did not results in increased bacterialclearance^([68-72]). Three doses at 14-day intervals were used in ourstudy because a similar schedule was reportedly efficacious when killedmycoplasmal bacterin was administered as an aerosol inchicken^([68-72]). The frequency of vaccination may depend on theefficiency of the clearance mechanism (mucociliary apparatus). Thegreater the efficiency, the greater the frequency to insure continuousexposure of the APT3Cooc10 with the M.-hyopneumoniae.

The appropriate dose for aerosol vaccination with APT3Cooc10 can bedetermined, for example, by determining the dose per cubic meter of airspace (mole/m³-air or Kg/m³-air) within the chamber or bam-space intowhich the pigs are confined for 30 to 45 minutes. To assess the properdosage of aptamers in aerosol (mole/mi-air), one may take into accountthe respiratory tract volume of the pigs (depends on the age), the lossassociated with the mucosal clearance mechanisms (depends on the age andthe gaseous environment), the loss associated with the particle sizedistribution (not only the Mass Median Average Diameter/MMAD) of thenebulized particles, and the loss average loss due to condensation, forexample.

TABLE 3 Mean O.D. (M. Hyopneumoniae antibody) for serum from pigsvaccinated by APT3 and CV injection. Control pigs were sham-vaccinatedwith nebulized PBS. Pre-vaccination Post-vaccination Post-challengeGroup # pigs Serum IgG Serum IgG Serum IgG PBS 7 0.397 ± 0.287 0.166 ±0.089 0.759 ± 0.217 CV 7 0.320 ± 0.259 0.134 ± 0.053 1.189^(b) ± 0.256 APT3 7 0.419 ± 0.286 0.313 ± 0.156 0.663 ± 0.353 ^(b)Significantlydifferent from the PBS-control group with α = 0.05 (Scheffé's method)

REFERENCES

-   ¹R. Desrosiers, Transmission of swine pathogens: Different means,    different needs. Animal Health Res. Rev, 2011, vol. 12, pp. 1-13.    Also available at the pigsite.com website. Also, P. Whittleston.    Porcine Mycoplasmas, pp. 133-166. In The Mycoplasmas, vol. II, 1970,    pp. 133-166, Ed. J. G. Tully and R. F. Whitcomb, Academic Press,    Inc., NY. Also Etiology and epidemiology, in ENZOOTIC PNEUMONIA: THE    DISEASE, produced by Hipra Inc. Also see the hipra.com website.-   ²M. Tajima &T. Yagihasi. Interaction of Mycoplasma hyopneumoniae    with the Porcine Respiratory Epithelium as Observed by    Electron-Microscopy. Infection and Immunity, 1982, vol. 37, pp.    1162-1169.-   ³C. Deblanc et al., Pre-infection of pigs with Mycoplasma    hyopneumoniae modifies outcomes of infection with European swine    influenza virus of H1N1, but not H1N2 subtype. Veterinary    Microbiology, 2012, vol. 157, pp. 96-105.-   ⁴E. L. Thacker et al., Mycoplasma hyopneumoniae: Potentiation or    Porcine Reproductive and Respiratory Syndrome Virus-induced    Pneumonia, J. of Clin. Microbiol., 1999, vol. 37, pp. 620-627.    Also, H. Nathues et al. Occurrence or Mycoplasma hyopneumoniae    infections in suckling and nursery pigs in region of high pig    density. Veterinary Records, 2010, vol. 166, pp. 194-198.-   ⁵T. Meyuns et al., Quantification of the spread of Mycoplasma    hyopneumoniae in nursery pigs using transmission experiments, Prev.    Vet. Med., 2004, vol. 66, pp. 265-275. Also see L. K. Clark, et al.,    Investigating the transmission or Mycoplasma hyopneumoniae in a    swine herd with enzootic pneumonia. Veterinary Medicine, 1991, vol.    86, pp. 543-550.-   ⁶K. D. C. Stark. Epidemiological investigation of the Influence of    environmental risk Factors on respiratory diseases in Swine—A    literature review. Veterinary J., 2000, vol. 159, pp. 37-56.    Also F. C. Minion, Molecular pathogenesis or Mycoplasma animal    respiratory pathogens. Frontiers in Bioscience, 2002, vol. 7, pp.    1410-1422. Also S. Otake et al. Long-distance transport of    infectious PRRSV and Mycoplasma hyopneumoniae from a swine    population infected with multiple viral variants. Vet. Microbiol.,    2010, vol. 145, pp. 198-208.-   ⁷P. Thongkamkoon et al. In Vitro susceptibility of Mycoplasma    hyopneumoniae field isolates and occurrence of Fluoroquinolone,    Macrolides and Lincomycin resistance. J. Vet. Med. Sci., 2013, vol.    75(8), pp. 1067-1070. Also L. L. Carron et al., Persistence of    Mycoplasma hyopneumoniae in experimentally infected pigs after    Marboflooxacin treatment and detection of mutation in the parC Gene,    Antimicrobial, agents and Chemotherapy, 2006, vol. 50, no. 6, pp.    1959-1966. Also J. et al. Resistance mechanism against    fluoroquinolones in Mycoplasma hyopneumoniae field isolates.    Microbiol. Drug Resistance, 2007, vol. 13, pp. 166-170.-   ⁸S. Beutinger et al., Local and systemic immune responses in pigs    intramuscularly injected with an inactivated Mycoplasma    hyopneumoniae vaccine. Vaccine, 2013, vol. 31, pp. 1305-1311. S.    Wilson et al., Vaccination of piglets at 1 week of age with an    Inactivated Mycoplasma hyopneumoniae vaccine reduces lung lesions    and improves average daily gain in body weight. Vaccine, 2012, vol.    30, pp. 7625-7629. P. D. Tassis et al., Clinical evaluation of    intradermal vaccination against porcine enzootic pneumonia    (Mycoplasma hyopneumoniae), Veterinary Records, 2012, vol. 170, pp.    261-267. Also, M. Pieters et al. An Experimental model to evaluate    Mycoplasma hyopneumoniae transmission from asymptomatic carriers to    unvaccinated and vaccinated sentinel pigs, Can. J. Vet. Res, 2012,    vol. 74, pp. 157-160. Also, F. Haesebrouck, et al., Efficacy of    vaccines against bacterial diseases in swine: what can we expect?,    Vet. Microbiology, 2004, vol. 100, pp. 255-268. I. Villarreal et    al., Effect of challenge of pigs previously immunised with    inactivated vaccines containing homologous and heterologous    Mycoplasma hyopneumoniae strains. BMC Vet. Res., 2012, vol. 8, 5    pages. A. Elsbernd et al. A Review on the Impact of Mycoplasma    hyopneumoniae Vaccination on Average Daily Gain in Swine. Iowa State    University Animal Industry Report, (Leaflet R2672) 2012, 3 pages.-   ⁹E. L. Thacker, Mycoplasmal Diseases, in Diseases of Swine, 9^(th)    Ed., 2006, vol. 42, pp. 701-717.-   ¹⁰R. Erlinger, Glycosaminoglycans in porcine lung: An    ultrastructural study using cupromeronic blue. Cell Tissue Res.,    1995, vol. 281, pp. 473-483.-   ¹¹F. C. Minion et al., The genome sequence of Mycoplasma    hyopneumoniae strain 232 the agent of swine mycoplasmosis. J. of    Bacteriology, 2004, vol. 186, pp. 7123-7133. Also, A. T. Vasconcelos    et al. Swine and poultry pathogens: the complete genome sequences of    two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma    synoviae. J. of Bacteriology, 2005, vol. 187, pp. 5568-5577.-   ¹²T. Hsu, S. Artiushin, & F. C. Minion, Cloning and functional    analysis of P97 Swine cilium adhesin gene of Mycoplasma    hyopneumoniae J. of Bacteriology, 1997, vol. 179, pp. 1317-1323.    Also F. C. Minion et al., R1 region of P97 mediates adherence of    Mycoplasma hyopneumoniae to swine cilia. Infection and Immunity,    2000, vol. 68, pp. 3056-3060. Also S. P. Djordjevic et al.,    Proteolytic processing of the Mycoplasma hyopneumoniae cilium    adhesin. Infection and Immunity, 2004, vol. 72, pp. 2791-2802.-   ¹³D. Maes et al., Control of Mycoplasma hyopneumoniae infections in    Pigs, Veterinary Microbiology, 2008, vol. 126, pp. 297-309.    Also L. A. de Castro et al., Variable numbers of tandem amino acid    repeats in adhesion-related CDS products in Mycoplasma hyopneumoniae    strains. Vet. Microbio, 2006, vol. 116, pp. 258-269, Also A. T.    Deutscher et al., Mycoplasma hyopneumoniae surface proteins M.    hyopneumoniaep385 and M. hyopneumoniaep384 bind host cilia and    glycosaminoglycans and are endopreteolytically processed by    proteases that recognize different cleavage motifs. J. of Proteome    Res., 2012, vol. 11, pp. 1924-1936. Also J. Wilton et al., M.    hyopneumoniaep493 (P216) is a proteolytically processed, cilium and    heparin binding protein of Mycoplasma hyopneumoniae. Molecular    Microbiology, 2009, vol. 71, pp. 566-582. Also L. M. Seymour et    al., M. hyopneumoniaep182 (P102) binds fibronectin and contribute to    the recruitment or plasminogen to the Mycoplasma hyopneumoniae cell    surface. Cellular Microbiology, 2012, vol. 14, pp. 81-94. Also L. M.    Seymour et al., M. hyopneumoniaep107 is a member of the    multifunctional adhesin family of Mycoplasma hyopneumoniae. J. of    Biol. Chem., 2011, vol. 286, pp. 10097-110104 Also L. M. Seymour et    al., A processed multidomain Mycoplasma hyopneumoniae adhesin binds    fibronectin, plasminogen, and swine respiratory cilia. J. of Biol.    Chem., 2010, vol. 285, pp. 33971-33979. Also A. T. Deutscher et al.,    Repeat regions R1 and R2 in the P97 paralogue M. hyopneumoniaep271    of Mycoplasma hyopneumoniae bind heparin, fibronectin and porcine    cilia. Molecular Microbiology, 2010, vol. 78, pp. 444-458.    Also D. R. Bogema et al., Characterization of cleavage events in the    multifunctional cilium adhesin M. hyopneumoniaep684 (P146) reveals a    mechanism by which Mycoplasma hyopneumoniae regulates surface    topography. mBio, 2012, vol. 3(2), pp. e00282-11. Also D. R. Bogema    et al., Sequence TTKF↓QE defines the site of proteolytic cleavage    in M. hyopneumoniaep63 protein, a novel glycosaminoglycan and cilium    adhesin of Mycoplasma hyopneumoniae. J. of Biol. Chem., 2011, vol.    286, pp. 41217-41229. Also T. A. Burnett et al., P159 is    proteolytically processed, surface adhesin of Mycoplasma    hyopneumoniae: Defined domains of P159 bind heparin and promote    adherence to eukaryote cells. Molecular Microbiology, 2006, vol. 60,    pp. 669-686.-   ¹⁴T. Hsu & F. C. Minion, Identification of the Cilium Binding    Epitope of the Mycoplasma hyopneumoniae P97 Adhesin, Infection &    Immunity, 1998, vol. 66, pp. 4762-4766. Also Q. Zhang, T. F. Young,    and R. F. Ross, Identification and characterization of a Mycoplasma    hyopneumoniae Adhesin, Infection and Immunity, 1995, vol. 63, pp.    1013-1019.-   ¹⁵C. Jenkins et al., Two domains within the Mycoplasma hyopneumoniae    cilium adhesin bind heparin. Infection and Immunity, 2006, vol. 74,    pp. 481-487.-   ¹⁶Plasminogen and fibronectin are highly abundant multi-functional    glycoproteins that circulate in body fluids. They are deposited on    cell surfaces and are extra-cellular matrix components. Fibronectin    is also important in wound repair of respiratory epithelial cells    and plasminogen is present in airway of healthy pigs. See L. M.    Seymour et al., M. hyopneumoniaep182 (P102) binds fibronectin and    contribute to the recruitment of plasminogen to the Mycoplasma    hyopneumoniae cell surface Cellular Microbiology, 2012, vol. 14, pp.    81-94. Also see M. Swaisgood et al., Plasminogen is an important    regulator in the pathogenesis of a murine model of asthma. Am. J. of    Respir, Crit. Care Med. 2007, vol. 176, pp. 333-342. Also see C.    Coraux et al., Epithelial extra-cellular matrix interactions and    stem cells in airway epithelial regeneration. Proc. Am. Thoracic    Soc., 2008, vol. 5, pp. 689-694).-   ¹⁷GenBank accession number U50901. Protein id=“AAB47806.1”    (Available at the National Center for Biotechnology Information    (NCBI) website).-   ¹⁸S. B. Marchioro, S. Simionatto, et al., Production and    characterisation or recombinant transmembrane proteins from    Mycoplasma hyopneumoniae. Veterinary Microbiology, 2012, vol. 155,    pp. 44-52.-   ¹⁹Also A. T. Deutscher et al., Repeat regions R1 and R2 in the P97    paralogue M. hyopneumoniaep271 of Mycoplasma hyopneumoniae bind    heparin, fibronectin and porcine cilia. Molecular Microbiology,    2010, vol. 78, pp. 444-158.-   ²⁰Q. Zhang, T. F. Young, and R. F. Ross, Microtiter Plate Adherence    Assay and receptor analogs for Mycoplasma hyopneumoniae. Infection &    Immunity, 1994, vol, 62, pp. 1616-1622.-   ²¹I. Dobrescu et al., In vitro and ex-vivo analyses of co-infections    with swine PRRS and porcine reproductive and respiratory syndrome    viruses. Vet. Microbiol., 2014, vol. 169, pp. 18-32.-   ²²M. Sibila et al. Current perspectives on the diagnosis and    epidemiology of Mycoplasma hyopneumoniae infection. Vet. J., 2009,    vol. 181, pp. 221-231. Also E. L. Thacker, Diagnosis of Mycoplasma    hyopneumoniae. J of Swine Health & Prod., 2004, vol. 12, pp.    252-254. Also R. Desrosiers, A review of some aspects of the    epidemiology, diagnosis, and control of Mycoplasma hyopneumoniae    infections. J of Swine Health and Prod. 2001, vol. 9, pp. 233-237.-   ²³V. Sorensen et al., Mycoplasma hyopneumoniae infection in pigs:    Duration of the disease and evaluation of four diagnostic assays,    Vet. Microbiol., 1997, vol. 54, pp. 23-33-   ²⁴L. Moorkamp et al. Detection of respiratory pathogens in porcine    long tissue and lavage fluid. Vet. J., 2008, vol. 175, pp. 273-275.-   ²⁵K. T. Kurth et al. Use of a Mycoplasma hyopneumoniae nested    polymerase chain reaction test to determine the optimal sampling    sites in swine. J. of Veterinary Diagnostic Invest. 2002, vol. 14,    pp. 463-469.-   ²⁶M. Pieters & C. Pijoan, Detection of Mycoplasma hyopneumoniae DNA    in experimentally infected pip. In the Proc. of the 19^(th) Int. Pig    Veterinary Society (IPVS), Copenhagen, 2006, p. 209.-   ²⁷SciFinder, available at the cas.org website. Also P. Dua, S. Kim,    & D K Lee, Patents on SELEX and therapeutic aptamers, in Recent Pat.    DNA Gene Seq. 2008, vol. 2, pp. 172-186. Also S. Missailids & A.    Hardy. Aptamers as inhibitors of target proteins, in Expert Opinion    on Therapeutics Patents, 2009, vol. 19, pp. 1073-1082.-   ²⁸D. S. Wilson & J. W. Szostak. In vitro selection of functional    nucleic acids. Annual Rev. Biochem., 1999, vol. 68, pp. 611-647.    Also A. Cabiel et al. Methods to identify aptamers against cell    surface biomarkers. Pharmaceuticals, 2011, vol. 4, pp. 1216-1235    Also B. Hall et al. Design, synthesis, and amplification of DNA    pools for in vitro selection. Current Protocols in Molecular    Biology, 2009, vol. 88, pp. 24.2.1-24.2.27. Also S. Chandra & B.    Gopinath. Methods developed for SELEX Anal. Bioanal. Chem., 2007,    vol. 387, pp. 171-182.-   ²⁹G. Mayer. The chemical biology of aptamers: A Review. Angew. Chem.    Int. Ed., 2009, vol. 48, pp. 2672-2689.-   ³⁰S. Balamuragan et al. Surface immobilisation methods for aptamer    diagnostic applications. Anal. Bioanal. Chem., 2008, vol. 390, pp.    1009-1021. Also X. Fang & W. Tan. Aptamers generated from Cell-SELEX    for molecular medicine: A chemical biology approach, in Accounts of    Chem. Res., 2010, vol. 43, pp. 48-57. Also J. Liu et al. Recent    developments in protein and cell-targeted aptamer selection and    applications, in Curr. Medi. Chem., 2011, vol. 18, pp. 4117-4125.-   ³¹Y. Zhang et al. Tumor-targeted drug delivery with aptamers. Curr.    Med. Chem., 2011, vol. 18, pp. 4185-4194. Also A. D. Keefe et al.    Aptamers as therapeutics, Nature Reviews: Drug discoveries, 2010,    vol. 9 pp. 337-550. Also Z. Zhang et al. Nucleic acid aptamers in    human viral disease. Arch. of Immunol. Ther. Exp., 2004, vol. 52,    pp. 307-315.-   ³²W. Zhao et al. Cell-surface sensors for real-time probing of    cellular environments, in Nature Nanotechnology, 2011, vol. 6, pp.    524-531. Also K. Sefah et al. Nucleic add aptamers for biosensors    and bio-analytical applications, Analyst, 2009, vol. 134, pp.    1765-1775. Also E. Torres-Chavolla & E. C. Alocilja. Aptasensors for    detection of microbial and viral pathogens. Biosensors & Bioelect,    2009, vol. 24, pp. 3173-3182. Also M. N. Velasco-Garcia & S.    Missailidis. New trends in aptamer-based electrochemical biosensors.    Gene Therapy & Mole. Biol., 2009, vol. 13, pp. 1-9. Also Y. Xiao et    al. Preparation of electrode-immobilized, redox-modified    oligonucleotides for electrochemical DNA and aptamer-based sensing.    Nature Protocols, 2007, vol. 2, pp. 2875-2880. I. Willner and M.    Zayats. Electronic Aptamer-Based Sensors Agnew. Chem., Int. Ed.,    2007, vol. 46, pp. 6408-6418. Also S. Song et. al. Aptamer-based    biosensors. Trends in Anal. Chem., 2008, vol. 27, pp. 108-117.-   ³³D. G. Burch, Controlling Mycoplasmal (Enzootic) Pneumonia, in in    Farmers Guide, UK, February 2004. Also D. Maes, Mycoplasma    hyopneumoniae infections in pigs: Update on epidemiology and    control, in Proc. of the 21^(st) IPVS Congress, Vancouver, 2010, pp.    30-35.-   ³⁴Mypravac Suis® (Hipra, Spain), Porcilis® (Merk Animal Health,    MycoFlex® (Boerhinger Ingelheim), Suvaxyn M. hyopneumoniae-One®    (Zoetis), etc-   ³⁵D. G. Burch, Mycoplasma vaccines—Comparative studies, in Pig World    Magazine, U K, 2001, (Available at the octagon-services.co.uk    website).-   ³⁶D. Maes et al. Effect of vaccination against Mycoplasma    hyopneumoniae in pig herds with an all-in/all-out production system.    Vaccine, 1999, vol. 17, pp. 1024-1034.-   ³⁷J. Vicca et al. In Vitro Susceptibilities of Mycoplasma    hyopneumoniae Field Isolates, Antimicrobial Agents & Chemotherapy,    2004, vol. 48, pp. 4470-4472. Also Inamoto et al., 1994-   ³⁸T. Meyns et al., Comparison of transmission of Mycoplasma    hyopneumoniae in vaccinated and non-vaccinated populations, Vaccine,    2006, vol. 24, pp. 7981-7086.-   ³⁹M. Sibila et al, Chronological study of Mycoplasma hyopneumoniae    infection, sero-conversion and associated lung lesions in vaccinated    and non-vaccinated pigs, Vet. Microbiol., 2007, vol. 122, pp.    197-201.-   ⁴⁰I. Villarreal et al., The effect of vaccination on the    transmission of Mycoplasma hyopneumoniae in pigs under field    conditions, Veterinary Journal, 2011, vol. 188 pp. 48-52.-   ⁴¹E. Roulet et al., High-throughput SELEX-SAGE method for    quantitative modeling of transcription-factor. Nature Biotechnology,    2002, vol. 20, pp. 831-835. Also A. B. Iliuk et al., Aptamers in    bioanalytical applications. Anal. Chem., 2011, vol. 83, pp.    4440-4452.-   ⁴²T. F. Young et al. A tissue culture system to study respiratory    ciliary epithelial adherence of selected swine mycoplasmas Vet.    Microbiol, 2000, vol. 71, pp. 269-279.-   ⁴³G. C. Zielinski & R. F. Ross, Adherence of Mycoplasma    hyopneumoniae to porcine ciliated respiratory tract cells. Am. J. of    Vet. Res., 1993, vol. 54, pp. 1262-1269.-   ⁴⁴M. DeBey & R. F. Ross, Ciliostasis and loss of cilia induced by    Mycoplasma hyopneumoniae in porcine tracheal organ cultures.    Infection & Immunity, 1994, vol. 62, pp. 5312-5318.-   ⁴⁵D. Calus et. al. In vivo virulence of Mycoplasma hyopneumoniae    Isolates does not correlate with in vitro adhesion assessed by a    microtitre plate adherence assay. J. of Appl. Microbiol., 2009, vol.    106, pp. 1951-1956.-   ⁴⁶M. Yamaya et al., Differentiated structure and function of    cultures from human tracheal epithelium. Am. J. of Physiology: Lung    & Cell. Mol. Physiol., 1992, vol. 262, no. 6, pp. L713-L724-   ⁴⁷A. B. Clarke et al., Regulation of ciliated cell differentiation    in cultures of rat tracheal epithelial cells. Am. J. of Resp. Cell &    Mole. Biol., 1995, vol. 12, pp. 329-338.-   ⁴⁸A. D. Keefe & S. T. Cload, SELEX with modified nucleotides. Curr.    Opinion in Chem. Biol., 2008, vol. 12, pp. 448-456. Also J. Wang    & G. Li. Aptamers against cell surface receptors: Selection,    modifications and application. Curr. Medi. Chem., 2011, vol. 18, pp.    4107-4116. R. E. Wang et al. Improving the stability of aptamers by    chemical modifications. Curr. Medi. Chem., 2011, vol. 18, pp.    4126-4138.-   ⁴⁹ The Concise Encyclopedia of Polymer Science And Engineering, pp.    858-859. J. I. Kroschwitz, ed. By John Wiley & Sons, 1990. Also U.    English & D. H. Gausset, Chemically Modified Oligonucleotides as    Probes and Inhibitors. Angewandte Chemie, Int. Edition, 1991, vol.    30, pp. 613-722. Also Y. S. Sanghvi, Chapter 15, Antisense Research    and Applications, pp. 273-288, ed. by S. T. Crooke and B. Lebleu,    published by CRC Press, Boca Raton, 1993.-   ⁵⁰Y. S. Sanghvi, Chapter 15, Antisense Research and Applications,    see pp. 276-278. Ed. by S. T. Crooke and B. Lebleu, published by CRC    Press, Boca Raton, 1993.-   ⁵¹See U.S. Pat. No. 6,303,374 (Antisense modulation of caspase 3    expression).-   ⁵²F. Szoka & D. Papahadjopoulos. Comparative properties and methods    of preparation of lipid vesicles (liposomes). Annual review of    biophysics and bioengineering, 1980, vol. 9, pp. 467-508. Also A.    Gomezhens & J. Fernandezromero. Analytical methods for the control    of liposomal delivery systems. TrAC Trends in Analytical Chemistry,    2006, vol. 25, pp. 167-178. Also M. R. Mozafari. Liposomes: an    overview of manufacturing techniques. Cell Mol Biol Lett., 2005,    vol. 10, pp, 711-719.-   ⁵³A. Jesorka & O. Orwar, Liposomes: Technologies and Analytical    Applications in Annual Rev. Anal. Chem., 2008, vol. 1, pp. 801-832.    Also B. Maherani et al. Liposomes: A Review of Manufacturing    Techniques and Targeting Strategies, in Current Nanoscience, 2011,    vol. 7, pp. 434-452.-   ⁵⁴see Remington's Pharmaceutical Sciences, Mack Publishing Co.,    Easton, Pa., latest edition. Also Advanced Drug Delivery:    Perspectives and prospects. In Advanced Drug Delivery Reviews, 2013,    vol. 65, no 1, pp. 1-148. Also see Drug delivery (Available at the    wikipedia.org website). Also see Drug Delivery Journal. Copyright by    Informa Healthcare USA, Inc. ISSN 1071-7544 print/ISSN 1521-0464    online. Also J. S. Patil & S. Sarasija. Pulmonary drug delivery    strategies: A concise, systematic review. Lung India, 2012, vol. 29,    pp. 44-49.-   ⁵⁵W. Rojanarat et al. Inhaled pyrazinamide proliposome for targeting    alveolar macrophages. Drug Delivery, 2012, vol. 19, pp. 334-345.-   ⁵⁶S. Parasuraman, Toxicological screening. J. of Pharmaco &    Pharmacotherapeutics, 2011, vol. 2(2), pp. 74-79. Also available at    the fda.gov website.-   ⁵⁷A. Noonberg et al., Nucleic Acids Res, 1994, vol. 22, pp.    2830-2836. L. Thompson et al., Nucleic Acids Res, 1995, vol. 23, pp.    2259-2268.-   ⁵⁸A. Kunkel & Pederson, Nucleic Acids Res., 1989, vol. 17, pp.    7371-7379. Also Kunkel et al., Proc. Natl. Acad Sci. USA, 1986, vol.    83, pp. 8575-8579. Also Reddy et al., J. Biol. Chem., 1987, vol.    262, pp. 75-81.-   ⁵⁹Hall et al., Cell, 1982, vol. 29, pp. 3-5. Also Nielsen et al.,    Nucleic Acids Res., 1993, vol. 21, pp. 3631-3636. Fowlkes & Shenk,    Cell, 1980, vol. 22, pp. 405-413. Also Gupta & Reddy, Nucleic Acids    Res., 1991, vol. 19, pp. 2073-2075. Kickhoefer et al., J. Biol.    Chem., 1993, vol. 268, pp. 7868-7873. Romero & Blackburn, Cell,    1991, vol. 67, pp. 343-333.-   ⁶⁰L. M. Khachigian. DNAzymes: cutting a path to a new class of    therapeutics. Curr. Opin. Mol. Ther., 2002, vol. 4, pp. 119-121.-   ⁶¹https://en.wikipedia.org/wiki/Fluorescent_tag#Methods_for_tracking_biomolecules-   ⁶² PCR Protocols: A Guide to Methods and Applications. Editors M. A.    Innis, D. H. Gelfand, J. J. Sninsky, & T. J. White. Academic Press,    1990.-   ⁶³see Rational Drug Design: Novel Methodology and Practical    Application, Editors A. L. Parrill & M. R. Reddy, Oxford University    Press, 1999. Also Advances In Antiviral Drug Design, Editor De    Clerq. Elsevier Science & Tech. 2007. Also Textbook of Drug Design    and Discovery. Editors U. Madsen, P. Krogsgaard-Larsen & T.    Liljefors. Taylor & Francis, 2002. ISBN 0-415-28288.-   ⁶⁴V. Lounnas et al. Current progress in Structure-Based Rational    Drug Design marks a new mindset in drug discovery, Compu. & Struc.    Biotech. J., 2013, vol. 5, e201302011. Also-   ⁶⁵http://en.wikipedia.org/wiki/Rational_drug_design-   ⁶⁶Aptamers specific for biomolecules and methods of making (U.S.    Pat. No. 3,756,291). Also Affinity Chromatography, Editor H. Schott,    Marcel Dekker, Inc., New York, 1984.-   ⁶⁷ Current Protocols in Molecular Biology, ed. by F. M. Ausubel et    al. John Wiley & Sons, 1999-2014 (DOI: 10.1002/0471142727).-   ⁶⁸D. A. Murphy et al., Aerosol vaccination of pigs against    Mycoplasma hyopneumoniae infection. Am. J. Vet. Res., 1993, vol.    54(11), pp 1874-1880.-   ⁶⁹A. T. M. Bosch et al., Viral and Bacterial Interactions In the    Upper Respiratory Tract. PLoS Pathog, 2013, vol. 9(1), paper    e1003057. Also R. J. Boynton and P. J. Oppenshow, Pulmonary Defenses    to acute respiratory infections. Brit. Med. Bull., 2002, vol. 61(1),    pp. 1-12. Also G. A. Laurenzi et al., Clearance of bacteria in the    lower respiratory, Science, 1963, vol. 142, pp. 1572-1573.-   ⁷⁰D. M. H. Hameleers et al., Mucosal and systemic antibody formation    in the rat after intranasal administration of three different    antigens. Immuno. Cell. Biol., 1991, vol. vol. 69, pp. 119-125.-   ⁷¹S. Morgan et al., Aerosol Delivery of a Candidate Universal    Influenza Vaccine Reduces Viral Load in Pigs Challenged with    Pandemic H1N1 Virus. J. of Immunology, 2016 (May),    doi:10.4049/jimmunol.1502632-   ⁷²E. Hayatsu et al., Local immunization in chicken respiratory tract    with killed Mycoplasma Gallisepticum vaccine. Japan J. of Vet. Sci.,    1974, vol. 36, pp. 311-319.

We claim:
 1. A nucleic acid molecule comprising: at least onepolynucleotide sequence aptamer capable of binding at least one portionof P97, P102, or P116 surface proteins on M. hyopneumoniae, wherein theat least one polynucleotide sequence aptamer is selected from the groupconsisting of: APT3Cooc10 (SEQ ID NO: 1), APT3Cooc4 (SEQ ID NO: 2),APT3Cooc2 (SEQ ID NO: 3), APT3Histo1 (SEQ ID NO: 4), APT3Cooc3 (SEQ IDNO: 5), APT3Histo2 (SEQ ID NO: 6), APT3Histo4 (SEQ ID NO: 7), APT3Histo5(SEQ ID NO: 8), APT3Histo9 (SEQ ID NO: 9), APT3Cooc7 (SEQ ID NO: 10),and combinations thereof, and wherein the at least one portion isselected from the group consisting of: amino acid positions 768 to 1082of the P97 surface protein (SEQ ID NO: 12), amino acid positions 768 to1050 of the P97 surface protein (SEQ ID NO: 24), amino acid positions768 to 925 of the P97 surface protein (SEQ ID NO: 13), amino acidpositions 1 to 529 of the P102 surface protein (SEQ ID NO: 25), aminoacid positions 1 to 324 of the P102 surface protein (SEQ ID NO: 26),amino acid positions 651 to 1010 of the P116 surface protein (SEQ ID NO:16), and combinations thereof.
 2. The nucleic acid molecule of claim 1,wherein the at least one polynucleotide sequence aptamer is furthercapable of binding at least one fragment having at least 80% identitywith the at least one portion.
 3. A nucleic acid molecule comprising: anaptamer capable of binding P97_({535-1028}) surface protein (SEQ ID NO:27) on M. hyopneumoniae, wherein the aptamer is selected from the groupconsisting of: APT3Cooc10 (SEQ ID NO: 1), APT3Cooc4 (SEQ ID NO: 2),APT3Cooc2 (SEQ ID NO: 3), APT3Histo1 (SEQ ID NO: 4), APT3Cooc3 (SEQ IDNO: 5), APT3Histo2 (SEQ ID NO: 6), APT3Histo4 (SEQ ID NO: 7), APT3Histo5(SEQ ID NO: 8), APT3Histo9 (SEQ ID NO: 9), APT3Cooc7 (SEQ ID NO: 10),and combinations thereof.
 4. The nucleic acid molecule of claim 3,wherein the aptamer is further capable of binding one or more repeatmotifs on P102 surface proteins on M. hyopneumoniae.
 5. A nucleic acidmolecule comprising: a single stranded DNA aptamer capable of bindingP97_({768-1082}) (SEQ ID NO: 12) and/or P97_({768-925}) (SEQ ID NO: 13)surface proteins on M. hyopneumoniae, wherein the aptamer is selectedfrom the group consisting of: APT3Cooc10 (SEQ ID NO: 1), APT3Cooc4 (SEQID NO: 2), APT3Cooc2 (SEQ ID NO: 3), APT3Histo1 (SEQ ID NO: 4),APT3Cooc3 (SEQ ID NO: 5), APT3Histo2 (SEQ ID NO: 6), APT3Histo4 (SEQ IDNO: 7), APT3Histo5 (SEQ ID NO: 8), APT3Histo9 (SEQ ID NO: 9), APT3Cooc7(SEQ ID NO: 10), and combinations thereof.
 6. The nucleic acid moleculeof claim 5, wherein the aptamer is further capable of binding one ormore repeat motifs on P102 surface proteins on M. hyopneumoniae.
 7. Amethod of identifying M. hyopneumoniae in a biological sample by bindingone or more surface proteins of M. hyopneumoniae, the method comprising:contacting the nucleic acid molecule of claim 1 with a biological samplecontaining M. hyopneumoniae so the nucleic acid molecule of claim 1binds with one or more surface proteins of the M. hyopneumoniae, therebyidentifying the M. hyopneumoniae in the biological sample.
 8. A methodof identifying M. hyopneumoniae in a biological sample by binding one ormore surface proteins of M. hyopneumoniae, the method comprising:contacting the nucleic acid molecule of claim 3 with a biological samplecontaining M. hyopneumoniae so the nucleic acid molecule of claim 3binds with one or more surface proteins of the M. hyopneumoniae, therebyidentifying the M. hyopneumoniae in the biological sample.
 9. A methodof identifying M. hyopneumoniae in a biological sample by binding one ormore surface proteins of M. hyopneumoniae, the method comprising:contacting the nucleic acid molecule of claim 5 with a biological samplecontaining M. hyopneumoniae so the nucleic acid molecule of claim 5binds with one or more surface proteins of the M. hyopneumoniae, therebyidentifying the M. hyopneumoniae in the biological sample.
 10. A methodof treating M. hyopneumoniae infection in a subject by binding one ormore surface proteins of M. hyopneumoniae, the method comprising:administering the nucleic acid molecule of claim 1 into a subjectinfected with M. hyopneumoniae so the nucleic acid molecule of claim 1binds with the M. hyopneumoniae present in the subject, thereby treatingthe M. hyopneumoniae infection.
 11. A method of treating M.hyopneumoniae infection in a subject by binding one or more surfaceproteins of M. hyopneumoniae, the method comprising: administering thenucleic acid molecule of claim 3 into a subject infected with M.hyopneumoniae so the nucleic acid molecule of claim 3 binds with the M.hyopneumoniae present in the subject, thereby treating the M.hyopneumoniae infection.
 12. A method of treating M. hyopneumoniaeinfection in a subject by binding one or more surface proteins of M.hyopneumoniae, the method comprising: administering the nucleic acidmolecule of claim 5 into a subject infected with M. hyopneumoniae so thenucleic acid molecule of claim 5 binds with the M. hyopneumoniae presentin the subject, thereby treating the M. hyopneumoniae infection.
 13. Acomposition comprising the nucleic acid molecule of claim 1 and one ormore additional agents, wherein at least one of the one or moreadditional agents comprises a carrier.
 14. A composition comprising thenucleic acid molecule of claim 3 one or more additional agents, whereinat least one of the one or more additional agents comprises a carrier.15. A composition comprising the nucleic acid molecule of claim 5 one ormore additional agents, wherein at least one of the one or moreadditional agents comprises a carrier.